AML

AML cells in the bone marrow

PHILADELPHIA—Preclinical research suggests a novel agent has preferential activity in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations.

The agent, VNLG-152, proved more cytotoxic in AML cell lines and patient samples with FLT3-ITD than in samples and cell lines with wild-type FLT3.

Exactly how and why this occurs remains somewhat of a mystery, however.

Sheetal Karne, MD, of the University of Maryland School of Medicine in Baltimore, and her colleagues detailed this mystery in a poster presentation at the AACR Annual Meeting 2015 (abstract 5408*).

Dr Karne noted that VNLG-152 targets translation by promoting the degradation of MAPK-interacting kinases (Mnks).

“[VNLG-152] has been previously published as functioning in Mnk degradation, which has been shown in triple-negative breast cancer and prostate cancer—in vivo and in vitro,” she said. “Our hypothesis was that, since [the drug] worked via decreasing translation, it would function in leukemia cells and, specifically, in leukemic cells with ITD mutations.”

So the investigators tested VNLG-152 in samples from AML patients, as well as both murine and human cell lines. They found that VNLG-152 was more cytotoxic in the presence of FLT3-ITD mutations, as evidenced by low micromolar IC₅₀ concentrations.

The IC₅₀ concentration was 3.4 μM in Ba/F3-ITD cells and 5.8 μM in Ba/F3-WT cells, which are murine cells transfected with human FLT3-ITD and wild-type FLT3, respectively. Similarly, the IC₅₀ concentration was 1.8 μM in 32D-ITD cells and 18.2 μM in 32D-WT cells.

In the human FLT3-ITD AML cell lines MV4-11 and MOLM-14, IC₅₀ concentrations were 2.3 μM and 4.2 μM, respectively. But concentrations were greater than 10 µM in the wild-type FLT3 human cell lines HL60 and U937.

In patient samples, the IC₅₀ concentration was 1.0 μM in FLT3-ITD AML and 7.5 μM in AML with wild-type FLT3.

In additional tests with murine cell lines, the investigators found that VNLG-152 inhibits the growth of Ba/F3-ITD and Ba/F3-WT cells. But the drug induces apoptosis in these cell lines only when given in high concentrations.

Looking into the mechanism of VNLG-152, the investigators found that the drug decreased Mnk-1 expression in Ba/F3-ITD and Ba/F3-WT cell lines.

“We saw that VNLG-152 worked via degradation of Mnk, but it was the same in both wild-type and ITD, so we’re still looking for an explanation as to what caused this difference,” Dr Karne said.

She and her colleagues believe the Mnk degradation inhibits the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), a downstream target of FLT3-ITD. But they are still investigating that possibility.

The team is also hoping to test VNLG-152 in combination with other drugs, such as FLT3 inhibitors or chemotherapeutic agents.

*Information in the abstract differs from that presented at the meeting.

AML cells in the bone marrow

PHILADELPHIA—Preclinical research suggests a novel agent has preferential activity in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations.

The agent, VNLG-152, proved more cytotoxic in AML cell lines and patient samples with FLT3-ITD than in samples and cell lines with wild-type FLT3.

Exactly how and why this occurs remains somewhat of a mystery, however.

Sheetal Karne, MD, of the University of Maryland School of Medicine in Baltimore, and her colleagues detailed this mystery in a poster presentation at the AACR Annual Meeting 2015 (abstract 5408*).

Dr Karne noted that VNLG-152 targets translation by promoting the degradation of MAPK-interacting kinases (Mnks).

“[VNLG-152] has been previously published as functioning in Mnk degradation, which has been shown in triple-negative breast cancer and prostate cancer—in vivo and in vitro,” she said. “Our hypothesis was that, since [the drug] worked via decreasing translation, it would function in leukemia cells and, specifically, in leukemic cells with ITD mutations.”

So the investigators tested VNLG-152 in samples from AML patients, as well as both murine and human cell lines. They found that VNLG-152 was more cytotoxic in the presence of FLT3-ITD mutations, as evidenced by low micromolar IC₅₀ concentrations.

The IC₅₀ concentration was 3.4 μM in Ba/F3-ITD cells and 5.8 μM in Ba/F3-WT cells, which are murine cells transfected with human FLT3-ITD and wild-type FLT3, respectively. Similarly, the IC₅₀ concentration was 1.8 μM in 32D-ITD cells and 18.2 μM in 32D-WT cells.

In the human FLT3-ITD AML cell lines MV4-11 and MOLM-14, IC₅₀ concentrations were 2.3 μM and 4.2 μM, respectively. But concentrations were greater than 10 µM in the wild-type FLT3 human cell lines HL60 and U937.

In patient samples, the IC₅₀ concentration was 1.0 μM in FLT3-ITD AML and 7.5 μM in AML with wild-type FLT3.

In additional tests with murine cell lines, the investigators found that VNLG-152 inhibits the growth of Ba/F3-ITD and Ba/F3-WT cells. But the drug induces apoptosis in these cell lines only when given in high concentrations.

Looking into the mechanism of VNLG-152, the investigators found that the drug decreased Mnk-1 expression in Ba/F3-ITD and Ba/F3-WT cell lines.

“We saw that VNLG-152 worked via degradation of Mnk, but it was the same in both wild-type and ITD, so we’re still looking for an explanation as to what caused this difference,” Dr Karne said.

She and her colleagues believe the Mnk degradation inhibits the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), a downstream target of FLT3-ITD. But they are still investigating that possibility.

The team is also hoping to test VNLG-152 in combination with other drugs, such as FLT3 inhibitors or chemotherapeutic agents.

*Information in the abstract differs from that presented at the meeting.

AML cells in the bone marrow

PHILADELPHIA—Preclinical research suggests a novel agent has preferential activity in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations.

The agent, VNLG-152, proved more cytotoxic in AML cell lines and patient samples with FLT3-ITD than in samples and cell lines with wild-type FLT3.

Exactly how and why this occurs remains somewhat of a mystery, however.

Sheetal Karne, MD, of the University of Maryland School of Medicine in Baltimore, and her colleagues detailed this mystery in a poster presentation at the AACR Annual Meeting 2015 (abstract 5408*).

Dr Karne noted that VNLG-152 targets translation by promoting the degradation of MAPK-interacting kinases (Mnks).

“[VNLG-152] has been previously published as functioning in Mnk degradation, which has been shown in triple-negative breast cancer and prostate cancer—in vivo and in vitro,” she said. “Our hypothesis was that, since [the drug] worked via decreasing translation, it would function in leukemia cells and, specifically, in leukemic cells with ITD mutations.”

So the investigators tested VNLG-152 in samples from AML patients, as well as both murine and human cell lines. They found that VNLG-152 was more cytotoxic in the presence of FLT3-ITD mutations, as evidenced by low micromolar IC₅₀ concentrations.

The IC₅₀ concentration was 3.4 μM in Ba/F3-ITD cells and 5.8 μM in Ba/F3-WT cells, which are murine cells transfected with human FLT3-ITD and wild-type FLT3, respectively. Similarly, the IC₅₀ concentration was 1.8 μM in 32D-ITD cells and 18.2 μM in 32D-WT cells.

In the human FLT3-ITD AML cell lines MV4-11 and MOLM-14, IC₅₀ concentrations were 2.3 μM and 4.2 μM, respectively. But concentrations were greater than 10 µM in the wild-type FLT3 human cell lines HL60 and U937.

In patient samples, the IC₅₀ concentration was 1.0 μM in FLT3-ITD AML and 7.5 μM in AML with wild-type FLT3.

In additional tests with murine cell lines, the investigators found that VNLG-152 inhibits the growth of Ba/F3-ITD and Ba/F3-WT cells. But the drug induces apoptosis in these cell lines only when given in high concentrations.

Looking into the mechanism of VNLG-152, the investigators found that the drug decreased Mnk-1 expression in Ba/F3-ITD and Ba/F3-WT cell lines.

“We saw that VNLG-152 worked via degradation of Mnk, but it was the same in both wild-type and ITD, so we’re still looking for an explanation as to what caused this difference,” Dr Karne said.

She and her colleagues believe the Mnk degradation inhibits the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), a downstream target of FLT3-ITD. But they are still investigating that possibility.

The team is also hoping to test VNLG-152 in combination with other drugs, such as FLT3 inhibitors or chemotherapeutic agents.

*Information in the abstract differs from that presented at the meeting.

Chengcheng Zhang, PhD

Photo courtesy of UT

Southwestern Medical Center

Preclinical research suggests that certain receptors containing the immunoreceptor tyrosine-based inhibition motif (ITIM) are important for the development of acute myeloid leukemia (AML).

“Although counterintuitive, this result is consistent with the generally immune-suppressive and, thus, tumor-promoting roles of inhibitory receptors in the immune system,” said Chengcheng Zhang, PhD, of UT Southwestern Medical Center in Dallas, Texas.

“These findings suggest that blocking ITIM-receptor signaling in combination with conventional therapies may represent a novel strategy for AML treatment.”

Dr Zhang and his colleagues reported their findings in Nature Cell Biology.

The team focused mainly on an ITIM-containing receptor called LAIR1. They found that deleting LAIR1 abolished leukemia in several different mouse models, without affecting normal hematopoiesis.

The investigators also identified a pathway that sustains the survival and self-renewal of AML cells, the mechanism by which LAIR1 supports AML development.

They said LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signaling adaptor to recruit CAMK1 for activation of downstream CREB in AML cells. And the LAIR1–SHP-1–CAMK1–CREB pathway sustains AML stem cells.

So the investigators believe that inhibiting the signaling initiated by LAIR1 and other ITIM-containing receptors could help us treat AML more effectively.

“Our study suggests that current treatment options, including chemotherapy, may not efficiently target cancer stem cells because these inhibitory receptors enable the leukemia stem cells to survive conventional therapies, eventually resulting in tumor relapse,” Dr Zhang said.

“The blockade of ITIM-receptor signaling may prove to be a novel, effective strategy for elimination of leukemia stem cells and lead to complete remission in patients.”

Chengcheng Zhang, PhD

Photo courtesy of UT

Southwestern Medical Center

Preclinical research suggests that certain receptors containing the immunoreceptor tyrosine-based inhibition motif (ITIM) are important for the development of acute myeloid leukemia (AML).

“Although counterintuitive, this result is consistent with the generally immune-suppressive and, thus, tumor-promoting roles of inhibitory receptors in the immune system,” said Chengcheng Zhang, PhD, of UT Southwestern Medical Center in Dallas, Texas.

“These findings suggest that blocking ITIM-receptor signaling in combination with conventional therapies may represent a novel strategy for AML treatment.”

Dr Zhang and his colleagues reported their findings in Nature Cell Biology.

The team focused mainly on an ITIM-containing receptor called LAIR1. They found that deleting LAIR1 abolished leukemia in several different mouse models, without affecting normal hematopoiesis.

The investigators also identified a pathway that sustains the survival and self-renewal of AML cells, the mechanism by which LAIR1 supports AML development.

They said LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signaling adaptor to recruit CAMK1 for activation of downstream CREB in AML cells. And the LAIR1–SHP-1–CAMK1–CREB pathway sustains AML stem cells.

So the investigators believe that inhibiting the signaling initiated by LAIR1 and other ITIM-containing receptors could help us treat AML more effectively.

“Our study suggests that current treatment options, including chemotherapy, may not efficiently target cancer stem cells because these inhibitory receptors enable the leukemia stem cells to survive conventional therapies, eventually resulting in tumor relapse,” Dr Zhang said.

“The blockade of ITIM-receptor signaling may prove to be a novel, effective strategy for elimination of leukemia stem cells and lead to complete remission in patients.”

Chengcheng Zhang, PhD

Photo courtesy of UT

Southwestern Medical Center

Preclinical research suggests that certain receptors containing the immunoreceptor tyrosine-based inhibition motif (ITIM) are important for the development of acute myeloid leukemia (AML).

“Although counterintuitive, this result is consistent with the generally immune-suppressive and, thus, tumor-promoting roles of inhibitory receptors in the immune system,” said Chengcheng Zhang, PhD, of UT Southwestern Medical Center in Dallas, Texas.

“These findings suggest that blocking ITIM-receptor signaling in combination with conventional therapies may represent a novel strategy for AML treatment.”

Dr Zhang and his colleagues reported their findings in Nature Cell Biology.

The team focused mainly on an ITIM-containing receptor called LAIR1. They found that deleting LAIR1 abolished leukemia in several different mouse models, without affecting normal hematopoiesis.

The investigators also identified a pathway that sustains the survival and self-renewal of AML cells, the mechanism by which LAIR1 supports AML development.

They said LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signaling adaptor to recruit CAMK1 for activation of downstream CREB in AML cells. And the LAIR1–SHP-1–CAMK1–CREB pathway sustains AML stem cells.

So the investigators believe that inhibiting the signaling initiated by LAIR1 and other ITIM-containing receptors could help us treat AML more effectively.

“Our study suggests that current treatment options, including chemotherapy, may not efficiently target cancer stem cells because these inhibitory receptors enable the leukemia stem cells to survive conventional therapies, eventually resulting in tumor relapse,” Dr Zhang said.

“The blockade of ITIM-receptor signaling may prove to be a novel, effective strategy for elimination of leukemia stem cells and lead to complete remission in patients.”

 

 

AACR Annual Meeting 2015

 

PHILADELPHIA—A retrospective study has revealed factors that appear to influence a person’s susceptibility to Waldenström’s macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) and other malignancies.

Study investigators looked at patients diagnosed with WM or LPL over a 20-year period and found about a 50% excess of second primary cancers in this population.

The patients had a significantly increased risk of multiple hematologic and solid tumor malignancies, and a few of these malignancies had shared susceptibility factors with WM/LPL.

The investigators believe that identifying these factors may prove useful for determining genetic susceptibility to WM/LPL.

Mary L. McMaster, MD, of the National Cancer Institute in Bethesda, Maryland, and her colleagues presented these findings at the AACR Annual Meeting 2015 (abstract 3709).

The team used data from the National Cancer Institute’s Surveillance, Epidemiology and End Results (SSER) database to evaluate the risk of subsequent primary cancer in 3825 patients diagnosed with WM (n=2163) or LPL (n=1662) from 1992 to 2011. The patients’ median age was 70, most of them were male (n=2221), and most were white (n=3153).

Dr McMaster said she and her colleagues looked at both WM and LPL in this study because SEER does not include information about immunoglobulin subtype, which makes it difficult to identify all WM cases with absolute certainty.

“[D]epending on what information a pathologist has when they review a bone marrow biopsy, for example, they may or may not know whether there’s IgM present,” Dr McMaster said. “So you may have a diagnosis of LPL and not have the information required to make the diagnosis of WM. For that reason, we combined both entities for this study.”

Dr McMaster and her colleagues calculated the observed-to-expected standardized incidence ratios (SIRs) for invasive cancers. After adjusting for multiple comparisons, the team found that survivors of WM/LPL had a significantly increased risk of developing a second primary malignancy (SIR=1.49).

This increased risk was seen for males and females and persisted throughout follow-up. The risk was higher for patients younger than 65 years of age (SIR=1.95).

Hematologic malignancies

WM/LPL survivors had a significantly increased risk of several hematologic malignancies. The SIR was 4.09 for all hematologic malignancies, 4.29 for lymphomas, and 3.16 for leukemias.

Dr McMaster pointed out that several lymphoma subtypes can have lymphoplasmacytic differentiation, the most common being marginal zone lymphoma. And this could potentially result in misclassification.

“So we actually ran the study with and without marginal zone lymphoma and saw no difference in the results,” she said. “So we don’t think misclassification accounts for the majority of what we’re seeing.”

The investigators found that WM/LPL survivors had the highest risk of developing Burkitt lymphoma (SIR=13.45), followed by Hodgkin lymphoma (SIR=9.80), T-cell non-Hodgkin lymphoma (SIR=6.62), mantle cell lymphoma (SIR=5.37), diffuse large B-cell lymphoma (DLBCL, SIR=4.76), multiple myeloma (SIR=4.40), any non-Hodgkin lymphoma (SIR=4.08), and acute myeloid leukemia (AML, SIR=3.27).

“Waldenström’s is known to transform, on occasion, to DLBCL,” Dr McMaster said. “So that may well account for the excess of DLBCL that we see in this population.”

She also noted that, prior to the early 2000s, WM was typically treated with alkylating agents. And alkylating agents have been linked to an increased risk of AML.

In this population, the risk of AML peaked 5 to 10 years after WM/LPL diagnosis and was only present in patients treated prior to 2002. This suggests the AML observed in this study was likely treatment-related.

Dr McMaster and her colleagues also found that WM/LPL survivors did not have a significantly increased risk of developing acute lymphocytic leukemia (SIR=0), hairy cell leukemia (SIR=0), chronic lymphocytic leukemia/small lymphocytic lymphoma (SIR=0.97), or follicular lymphoma (SIR=2.25).

Solid tumors

WM/LPL survivors did have a significantly increased risk of certain solid tumor malignancies. The overall SIR for solid tumors was 1.21.

The risk was significant for non-epithelial skin cancers (SIR=5.15), thyroid cancers (SIR=3.13), melanoma (SIR=1.72), and cancers of the lung and bronchus (SIR=1.44) or respiratory system (SIR=1.42).

“Melanoma has an immunological basis, as does Waldenström’s, so we think there may be some shared etiology there,” Dr McMaster said.

She also noted that a strong risk factor for thyroid cancer, particularly papillary thyroid cancer, is a history of autoimmune thyroid disease.

“Autoimmune disease of any sort is a risk factor for Waldenström’s macroglobulinemia,” she said. “So again, we think there might be a basis for shared susceptibility there.”

Dr McMaster said this research suggests that multiple primary cancers may occur in a single individual because of shared genetic susceptibility, shared environmental exposures, treatment effects, or chance. She believes future research will show that both genetic and environmental factors contribute to WM.

Investigators are currently conducting whole-exome sequencing studies and genome-wide association studies in patients with familial and spontaneous WM, with the hopes of identifying genes that contribute to WM susceptibility.

 

 

AACR Annual Meeting 2015

 

PHILADELPHIA—A retrospective study has revealed factors that appear to influence a person’s susceptibility to Waldenström’s macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) and other malignancies.

Study investigators looked at patients diagnosed with WM or LPL over a 20-year period and found about a 50% excess of second primary cancers in this population.

The patients had a significantly increased risk of multiple hematologic and solid tumor malignancies, and a few of these malignancies had shared susceptibility factors with WM/LPL.

The investigators believe that identifying these factors may prove useful for determining genetic susceptibility to WM/LPL.

Mary L. McMaster, MD, of the National Cancer Institute in Bethesda, Maryland, and her colleagues presented these findings at the AACR Annual Meeting 2015 (abstract 3709).

The team used data from the National Cancer Institute’s Surveillance, Epidemiology and End Results (SSER) database to evaluate the risk of subsequent primary cancer in 3825 patients diagnosed with WM (n=2163) or LPL (n=1662) from 1992 to 2011. The patients’ median age was 70, most of them were male (n=2221), and most were white (n=3153).

Dr McMaster said she and her colleagues looked at both WM and LPL in this study because SEER does not include information about immunoglobulin subtype, which makes it difficult to identify all WM cases with absolute certainty.

“[D]epending on what information a pathologist has when they review a bone marrow biopsy, for example, they may or may not know whether there’s IgM present,” Dr McMaster said. “So you may have a diagnosis of LPL and not have the information required to make the diagnosis of WM. For that reason, we combined both entities for this study.”

Dr McMaster and her colleagues calculated the observed-to-expected standardized incidence ratios (SIRs) for invasive cancers. After adjusting for multiple comparisons, the team found that survivors of WM/LPL had a significantly increased risk of developing a second primary malignancy (SIR=1.49).

This increased risk was seen for males and females and persisted throughout follow-up. The risk was higher for patients younger than 65 years of age (SIR=1.95).

Hematologic malignancies

WM/LPL survivors had a significantly increased risk of several hematologic malignancies. The SIR was 4.09 for all hematologic malignancies, 4.29 for lymphomas, and 3.16 for leukemias.

Dr McMaster pointed out that several lymphoma subtypes can have lymphoplasmacytic differentiation, the most common being marginal zone lymphoma. And this could potentially result in misclassification.

“So we actually ran the study with and without marginal zone lymphoma and saw no difference in the results,” she said. “So we don’t think misclassification accounts for the majority of what we’re seeing.”

The investigators found that WM/LPL survivors had the highest risk of developing Burkitt lymphoma (SIR=13.45), followed by Hodgkin lymphoma (SIR=9.80), T-cell non-Hodgkin lymphoma (SIR=6.62), mantle cell lymphoma (SIR=5.37), diffuse large B-cell lymphoma (DLBCL, SIR=4.76), multiple myeloma (SIR=4.40), any non-Hodgkin lymphoma (SIR=4.08), and acute myeloid leukemia (AML, SIR=3.27).

“Waldenström’s is known to transform, on occasion, to DLBCL,” Dr McMaster said. “So that may well account for the excess of DLBCL that we see in this population.”

She also noted that, prior to the early 2000s, WM was typically treated with alkylating agents. And alkylating agents have been linked to an increased risk of AML.

In this population, the risk of AML peaked 5 to 10 years after WM/LPL diagnosis and was only present in patients treated prior to 2002. This suggests the AML observed in this study was likely treatment-related.

Dr McMaster and her colleagues also found that WM/LPL survivors did not have a significantly increased risk of developing acute lymphocytic leukemia (SIR=0), hairy cell leukemia (SIR=0), chronic lymphocytic leukemia/small lymphocytic lymphoma (SIR=0.97), or follicular lymphoma (SIR=2.25).

Solid tumors

WM/LPL survivors did have a significantly increased risk of certain solid tumor malignancies. The overall SIR for solid tumors was 1.21.

The risk was significant for non-epithelial skin cancers (SIR=5.15), thyroid cancers (SIR=3.13), melanoma (SIR=1.72), and cancers of the lung and bronchus (SIR=1.44) or respiratory system (SIR=1.42).

“Melanoma has an immunological basis, as does Waldenström’s, so we think there may be some shared etiology there,” Dr McMaster said.

She also noted that a strong risk factor for thyroid cancer, particularly papillary thyroid cancer, is a history of autoimmune thyroid disease.

“Autoimmune disease of any sort is a risk factor for Waldenström’s macroglobulinemia,” she said. “So again, we think there might be a basis for shared susceptibility there.”

Dr McMaster said this research suggests that multiple primary cancers may occur in a single individual because of shared genetic susceptibility, shared environmental exposures, treatment effects, or chance. She believes future research will show that both genetic and environmental factors contribute to WM.

Investigators are currently conducting whole-exome sequencing studies and genome-wide association studies in patients with familial and spontaneous WM, with the hopes of identifying genes that contribute to WM susceptibility.

 

 

AACR Annual Meeting 2015

 

PHILADELPHIA—A retrospective study has revealed factors that appear to influence a person’s susceptibility to Waldenström’s macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) and other malignancies.

Study investigators looked at patients diagnosed with WM or LPL over a 20-year period and found about a 50% excess of second primary cancers in this population.

The patients had a significantly increased risk of multiple hematologic and solid tumor malignancies, and a few of these malignancies had shared susceptibility factors with WM/LPL.

The investigators believe that identifying these factors may prove useful for determining genetic susceptibility to WM/LPL.

Mary L. McMaster, MD, of the National Cancer Institute in Bethesda, Maryland, and her colleagues presented these findings at the AACR Annual Meeting 2015 (abstract 3709).

The team used data from the National Cancer Institute’s Surveillance, Epidemiology and End Results (SSER) database to evaluate the risk of subsequent primary cancer in 3825 patients diagnosed with WM (n=2163) or LPL (n=1662) from 1992 to 2011. The patients’ median age was 70, most of them were male (n=2221), and most were white (n=3153).

Dr McMaster said she and her colleagues looked at both WM and LPL in this study because SEER does not include information about immunoglobulin subtype, which makes it difficult to identify all WM cases with absolute certainty.

“[D]epending on what information a pathologist has when they review a bone marrow biopsy, for example, they may or may not know whether there’s IgM present,” Dr McMaster said. “So you may have a diagnosis of LPL and not have the information required to make the diagnosis of WM. For that reason, we combined both entities for this study.”

Dr McMaster and her colleagues calculated the observed-to-expected standardized incidence ratios (SIRs) for invasive cancers. After adjusting for multiple comparisons, the team found that survivors of WM/LPL had a significantly increased risk of developing a second primary malignancy (SIR=1.49).

This increased risk was seen for males and females and persisted throughout follow-up. The risk was higher for patients younger than 65 years of age (SIR=1.95).

Hematologic malignancies

WM/LPL survivors had a significantly increased risk of several hematologic malignancies. The SIR was 4.09 for all hematologic malignancies, 4.29 for lymphomas, and 3.16 for leukemias.

Dr McMaster pointed out that several lymphoma subtypes can have lymphoplasmacytic differentiation, the most common being marginal zone lymphoma. And this could potentially result in misclassification.

“So we actually ran the study with and without marginal zone lymphoma and saw no difference in the results,” she said. “So we don’t think misclassification accounts for the majority of what we’re seeing.”

The investigators found that WM/LPL survivors had the highest risk of developing Burkitt lymphoma (SIR=13.45), followed by Hodgkin lymphoma (SIR=9.80), T-cell non-Hodgkin lymphoma (SIR=6.62), mantle cell lymphoma (SIR=5.37), diffuse large B-cell lymphoma (DLBCL, SIR=4.76), multiple myeloma (SIR=4.40), any non-Hodgkin lymphoma (SIR=4.08), and acute myeloid leukemia (AML, SIR=3.27).

“Waldenström’s is known to transform, on occasion, to DLBCL,” Dr McMaster said. “So that may well account for the excess of DLBCL that we see in this population.”

She also noted that, prior to the early 2000s, WM was typically treated with alkylating agents. And alkylating agents have been linked to an increased risk of AML.

In this population, the risk of AML peaked 5 to 10 years after WM/LPL diagnosis and was only present in patients treated prior to 2002. This suggests the AML observed in this study was likely treatment-related.

Dr McMaster and her colleagues also found that WM/LPL survivors did not have a significantly increased risk of developing acute lymphocytic leukemia (SIR=0), hairy cell leukemia (SIR=0), chronic lymphocytic leukemia/small lymphocytic lymphoma (SIR=0.97), or follicular lymphoma (SIR=2.25).

Solid tumors

WM/LPL survivors did have a significantly increased risk of certain solid tumor malignancies. The overall SIR for solid tumors was 1.21.

The risk was significant for non-epithelial skin cancers (SIR=5.15), thyroid cancers (SIR=3.13), melanoma (SIR=1.72), and cancers of the lung and bronchus (SIR=1.44) or respiratory system (SIR=1.42).

“Melanoma has an immunological basis, as does Waldenström’s, so we think there may be some shared etiology there,” Dr McMaster said.

She also noted that a strong risk factor for thyroid cancer, particularly papillary thyroid cancer, is a history of autoimmune thyroid disease.

“Autoimmune disease of any sort is a risk factor for Waldenström’s macroglobulinemia,” she said. “So again, we think there might be a basis for shared susceptibility there.”

Dr McMaster said this research suggests that multiple primary cancers may occur in a single individual because of shared genetic susceptibility, shared environmental exposures, treatment effects, or chance. She believes future research will show that both genetic and environmental factors contribute to WM.

Investigators are currently conducting whole-exome sequencing studies and genome-wide association studies in patients with familial and spontaneous WM, with the hopes of identifying genes that contribute to WM susceptibility.

Lab mouse

PHILADELPHIA—Preclinical research suggests a cyclin-dependent kinase (CDK) inhibitor is active against acute leukemias, particularly those with mixed-lineage leukemia rearrangements (MLL-r).

CYC065 selectively inhibits CDK2, which drives cell-cycle transition and activates major DNA double-strand break repair pathways; CDK5, which drives metastatic spread; and CDK9, which regulates the transcription of genes important for the proliferation and survival of malignant cells.

Experiments have shown that CYC065 exhibits activity against acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), with and without MLL-r.

Daniella Zheleva, PhD, and her colleagues described these experiments in a poster at the AACR Annual Meeting 2015 (abstract 1650). All of the researchers are employees of Cyclacel Ltd., the company developing CYC065.

The researchers tested CYC065 in a panel of AML cell lines with wild-type MLL (HEL, HL60, Kasumi-1, KG-1, OCI-AML5, and PL21) and MLL-r (EOL-1, ML-2, MOLM-13, MV4-11, Nomo-1, OCI-AML2, and THP-1).

They found that MLL-r cell lines were “highly sensitive” to CYC065, but the sensitivity of cell lines with wild-type MLL correlated with the level of Bcl-2 family proteins. In the wild-type cell lines, IC_50/70/90 values were correlated with Bcl_XL and inversely correlated with Bak.

Six-hour pulse treatment of CYC065 at 0.5 µM to 1 µM was sufficient to cause 90% or greater cell death in sensitive cell lines. And cell lines with reduced sensitivity to the drug could be targeted by exposure to 10-hour pulse treatments of CYC065, or to CYC065 in combination with short pulses of Bcl-2 inhibitors.

The researchers observed “potent antitumor activity” when they administered CYC065 in AML xenograft models.

In an EOL-1 model, the median tumor growth inhibition on day 19 was 97% for mice that received CYC065 at 40 mg/kg (every day on days 1-5 and 8-12), 95% for mice that received CYC065 at 20 mg/kg every day on days 1-5 and 8-12), and 41% for mice that received cytarabine at 100 mg/kg (every day on days 1-5).

In the HL60 model, the median tumor growth inhibition on day 11 was 90% for mice that received CYC065 at 70 mg/kg (every day on days 1-5 and 8-12). And 2 mice achieved a complete response to treatment.

The researchers also found that CYC065 synergizes with cytarabine, particularly when CYC065 is given first. In fact, the combination could overcome the cytarabine resistance observed in the MV4-11 cell line.

CYC065 was “strongly synergistic” with Bcl2/Bcl_XL inhibitors as well, the researchers said. CYC065 synergized with ABT-199, ABT-263, and ABT-737 in both AML cell lines (THP-1 and HEL) and ALL cell lines (Jurkat and SEM).

The researchers said the potent in vitro and in vivo activity of CYC065 and the ability to combine the drug with other antileukemic agents suggest that it may have therapeutic potential in AML and ALL.

Lab mouse

PHILADELPHIA—Preclinical research suggests a cyclin-dependent kinase (CDK) inhibitor is active against acute leukemias, particularly those with mixed-lineage leukemia rearrangements (MLL-r).

CYC065 selectively inhibits CDK2, which drives cell-cycle transition and activates major DNA double-strand break repair pathways; CDK5, which drives metastatic spread; and CDK9, which regulates the transcription of genes important for the proliferation and survival of malignant cells.

Experiments have shown that CYC065 exhibits activity against acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), with and without MLL-r.

Daniella Zheleva, PhD, and her colleagues described these experiments in a poster at the AACR Annual Meeting 2015 (abstract 1650). All of the researchers are employees of Cyclacel Ltd., the company developing CYC065.

The researchers tested CYC065 in a panel of AML cell lines with wild-type MLL (HEL, HL60, Kasumi-1, KG-1, OCI-AML5, and PL21) and MLL-r (EOL-1, ML-2, MOLM-13, MV4-11, Nomo-1, OCI-AML2, and THP-1).

They found that MLL-r cell lines were “highly sensitive” to CYC065, but the sensitivity of cell lines with wild-type MLL correlated with the level of Bcl-2 family proteins. In the wild-type cell lines, IC_50/70/90 values were correlated with Bcl_XL and inversely correlated with Bak.

Six-hour pulse treatment of CYC065 at 0.5 µM to 1 µM was sufficient to cause 90% or greater cell death in sensitive cell lines. And cell lines with reduced sensitivity to the drug could be targeted by exposure to 10-hour pulse treatments of CYC065, or to CYC065 in combination with short pulses of Bcl-2 inhibitors.

The researchers observed “potent antitumor activity” when they administered CYC065 in AML xenograft models.

In an EOL-1 model, the median tumor growth inhibition on day 19 was 97% for mice that received CYC065 at 40 mg/kg (every day on days 1-5 and 8-12), 95% for mice that received CYC065 at 20 mg/kg every day on days 1-5 and 8-12), and 41% for mice that received cytarabine at 100 mg/kg (every day on days 1-5).

In the HL60 model, the median tumor growth inhibition on day 11 was 90% for mice that received CYC065 at 70 mg/kg (every day on days 1-5 and 8-12). And 2 mice achieved a complete response to treatment.

The researchers also found that CYC065 synergizes with cytarabine, particularly when CYC065 is given first. In fact, the combination could overcome the cytarabine resistance observed in the MV4-11 cell line.

CYC065 was “strongly synergistic” with Bcl2/Bcl_XL inhibitors as well, the researchers said. CYC065 synergized with ABT-199, ABT-263, and ABT-737 in both AML cell lines (THP-1 and HEL) and ALL cell lines (Jurkat and SEM).

The researchers said the potent in vitro and in vivo activity of CYC065 and the ability to combine the drug with other antileukemic agents suggest that it may have therapeutic potential in AML and ALL.

Lab mouse

PHILADELPHIA—Preclinical research suggests a cyclin-dependent kinase (CDK) inhibitor is active against acute leukemias, particularly those with mixed-lineage leukemia rearrangements (MLL-r).

CYC065 selectively inhibits CDK2, which drives cell-cycle transition and activates major DNA double-strand break repair pathways; CDK5, which drives metastatic spread; and CDK9, which regulates the transcription of genes important for the proliferation and survival of malignant cells.

Experiments have shown that CYC065 exhibits activity against acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), with and without MLL-r.

Daniella Zheleva, PhD, and her colleagues described these experiments in a poster at the AACR Annual Meeting 2015 (abstract 1650). All of the researchers are employees of Cyclacel Ltd., the company developing CYC065.

The researchers tested CYC065 in a panel of AML cell lines with wild-type MLL (HEL, HL60, Kasumi-1, KG-1, OCI-AML5, and PL21) and MLL-r (EOL-1, ML-2, MOLM-13, MV4-11, Nomo-1, OCI-AML2, and THP-1).

They found that MLL-r cell lines were “highly sensitive” to CYC065, but the sensitivity of cell lines with wild-type MLL correlated with the level of Bcl-2 family proteins. In the wild-type cell lines, IC_50/70/90 values were correlated with Bcl_XL and inversely correlated with Bak.

Six-hour pulse treatment of CYC065 at 0.5 µM to 1 µM was sufficient to cause 90% or greater cell death in sensitive cell lines. And cell lines with reduced sensitivity to the drug could be targeted by exposure to 10-hour pulse treatments of CYC065, or to CYC065 in combination with short pulses of Bcl-2 inhibitors.

The researchers observed “potent antitumor activity” when they administered CYC065 in AML xenograft models.

In an EOL-1 model, the median tumor growth inhibition on day 19 was 97% for mice that received CYC065 at 40 mg/kg (every day on days 1-5 and 8-12), 95% for mice that received CYC065 at 20 mg/kg every day on days 1-5 and 8-12), and 41% for mice that received cytarabine at 100 mg/kg (every day on days 1-5).

In the HL60 model, the median tumor growth inhibition on day 11 was 90% for mice that received CYC065 at 70 mg/kg (every day on days 1-5 and 8-12). And 2 mice achieved a complete response to treatment.

The researchers also found that CYC065 synergizes with cytarabine, particularly when CYC065 is given first. In fact, the combination could overcome the cytarabine resistance observed in the MV4-11 cell line.

CYC065 was “strongly synergistic” with Bcl2/Bcl_XL inhibitors as well, the researchers said. CYC065 synergized with ABT-199, ABT-263, and ABT-737 in both AML cell lines (THP-1 and HEL) and ALL cell lines (Jurkat and SEM).

The researchers said the potent in vitro and in vivo activity of CYC065 and the ability to combine the drug with other antileukemic agents suggest that it may have therapeutic potential in AML and ALL.

AML cells in the bone marrow

Results of single-cell genotyping suggest the evolution of acute myeloid leukemia (AML) is more complex than we thought.

In screening for mutations in 2 genes, researchers identified at least 9 distinct clonal populations, each harboring unique mutational patterns.

Some mutations seemed to arise not sequentially, but independently and at different points in time.

These results suggest single-cell analysis may be more effective than bulk-tumor analysis for assessing cancer evolution.

Carlo Maley, PhD, of Arizona State University in Tempe, and his colleagues recounted the results in Science Translational Medicine.

The researchers set out to provide a more accurate picture of what takes place at the genetic level when an AML patient experiences relapse or metastasis. So they examined individual cells, screening them for  mutations in FLT3 and NPM1.

The results suggested the same mutation was occurring multiple times within an AML patient. The team examined individual cells from 6 AML patients, and the results showed all combinations of homozygous and heterozygous mutations of FLT3 and NPM1.

“There’s no way to explain that with each mutation only happening once,” Dr Maley said. “That’s scary because it means that these cancers have access to many mutations and can find the same mutation over and over.”

Dr Maley noted that the process of convergent evolution, in which separate lineages develop similar features, appears to account for some of the observed diversity. And influences from the environment may drive convergent evolution, but identical mutations can also arise through pure coincidence, simply by virtue of the enormous numbers involved.

For example, a 1 cm³ AML tumor may contain a billion cells, each containing some 3 billion base pairs in its genome. Mutations are estimated to occur at a rate of 1 mutation in every billion base pairs.

“That means every time the population of cells in a 1 cm³ tumor undergoes 1 generation, which we think takes just a couple days, every possible mutation of the genome is happening somewhere in that tumor,” Dr Maley said.

This alone would lead to the same mutation likely occurring independently multiple times.

Curbing cancer’s lethality

Given AML’s near-limitless capacity for creating novel variants, what can clinicians do to halt the disease’s advance? According to Dr Maley, one approach would be to use cancer’s ability to evolve to our advantage, rather than attempt to fight it head on.

This paradigm draws on a branch of ecology known as life history theory. The idea is to carefully study the environmental factors that may lead organisms to favor either a fast or slow reproducing strategy to maximize their ability to survive.

According to the theory, fast reproduction tends to occur in environments with high extrinsic mortality. Aggressive cancer treatment creates just such an environment, favoring those cells able to reproduce quickly, producing large numbers of daughter cells, with a few evading extrinsic mortality to repopulate the tumor.

On the other hand, a very stable environment often favors slow reproduction, because organisms reach a carrying capacity of their surrounding environment. In this case, the limiting factor becomes competition between like organisms. Here, a slow reproducing strategy favoring greater investment in maintenance and survivability wins the competition.

“This approach would say, ‘Let’s keep tumors as stable as possible and keep their resources limited,’” Dr Maley said. “If we are able to keep the tumor cells contained and let them fight it out, we would expect to see more competitively fit cells that are growing very slowly.”

AML cells in the bone marrow

Results of single-cell genotyping suggest the evolution of acute myeloid leukemia (AML) is more complex than we thought.

In screening for mutations in 2 genes, researchers identified at least 9 distinct clonal populations, each harboring unique mutational patterns.

Some mutations seemed to arise not sequentially, but independently and at different points in time.

These results suggest single-cell analysis may be more effective than bulk-tumor analysis for assessing cancer evolution.

Carlo Maley, PhD, of Arizona State University in Tempe, and his colleagues recounted the results in Science Translational Medicine.

The researchers set out to provide a more accurate picture of what takes place at the genetic level when an AML patient experiences relapse or metastasis. So they examined individual cells, screening them for  mutations in FLT3 and NPM1.

The results suggested the same mutation was occurring multiple times within an AML patient. The team examined individual cells from 6 AML patients, and the results showed all combinations of homozygous and heterozygous mutations of FLT3 and NPM1.

“There’s no way to explain that with each mutation only happening once,” Dr Maley said. “That’s scary because it means that these cancers have access to many mutations and can find the same mutation over and over.”

Dr Maley noted that the process of convergent evolution, in which separate lineages develop similar features, appears to account for some of the observed diversity. And influences from the environment may drive convergent evolution, but identical mutations can also arise through pure coincidence, simply by virtue of the enormous numbers involved.

For example, a 1 cm³ AML tumor may contain a billion cells, each containing some 3 billion base pairs in its genome. Mutations are estimated to occur at a rate of 1 mutation in every billion base pairs.

“That means every time the population of cells in a 1 cm³ tumor undergoes 1 generation, which we think takes just a couple days, every possible mutation of the genome is happening somewhere in that tumor,” Dr Maley said.

This alone would lead to the same mutation likely occurring independently multiple times.

Curbing cancer’s lethality

Given AML’s near-limitless capacity for creating novel variants, what can clinicians do to halt the disease’s advance? According to Dr Maley, one approach would be to use cancer’s ability to evolve to our advantage, rather than attempt to fight it head on.

This paradigm draws on a branch of ecology known as life history theory. The idea is to carefully study the environmental factors that may lead organisms to favor either a fast or slow reproducing strategy to maximize their ability to survive.

According to the theory, fast reproduction tends to occur in environments with high extrinsic mortality. Aggressive cancer treatment creates just such an environment, favoring those cells able to reproduce quickly, producing large numbers of daughter cells, with a few evading extrinsic mortality to repopulate the tumor.

On the other hand, a very stable environment often favors slow reproduction, because organisms reach a carrying capacity of their surrounding environment. In this case, the limiting factor becomes competition between like organisms. Here, a slow reproducing strategy favoring greater investment in maintenance and survivability wins the competition.

“This approach would say, ‘Let’s keep tumors as stable as possible and keep their resources limited,’” Dr Maley said. “If we are able to keep the tumor cells contained and let them fight it out, we would expect to see more competitively fit cells that are growing very slowly.”

AML cells in the bone marrow

Results of single-cell genotyping suggest the evolution of acute myeloid leukemia (AML) is more complex than we thought.

In screening for mutations in 2 genes, researchers identified at least 9 distinct clonal populations, each harboring unique mutational patterns.

Some mutations seemed to arise not sequentially, but independently and at different points in time.

These results suggest single-cell analysis may be more effective than bulk-tumor analysis for assessing cancer evolution.

Carlo Maley, PhD, of Arizona State University in Tempe, and his colleagues recounted the results in Science Translational Medicine.

The researchers set out to provide a more accurate picture of what takes place at the genetic level when an AML patient experiences relapse or metastasis. So they examined individual cells, screening them for  mutations in FLT3 and NPM1.

The results suggested the same mutation was occurring multiple times within an AML patient. The team examined individual cells from 6 AML patients, and the results showed all combinations of homozygous and heterozygous mutations of FLT3 and NPM1.

“There’s no way to explain that with each mutation only happening once,” Dr Maley said. “That’s scary because it means that these cancers have access to many mutations and can find the same mutation over and over.”

Dr Maley noted that the process of convergent evolution, in which separate lineages develop similar features, appears to account for some of the observed diversity. And influences from the environment may drive convergent evolution, but identical mutations can also arise through pure coincidence, simply by virtue of the enormous numbers involved.

For example, a 1 cm³ AML tumor may contain a billion cells, each containing some 3 billion base pairs in its genome. Mutations are estimated to occur at a rate of 1 mutation in every billion base pairs.

“That means every time the population of cells in a 1 cm³ tumor undergoes 1 generation, which we think takes just a couple days, every possible mutation of the genome is happening somewhere in that tumor,” Dr Maley said.

This alone would lead to the same mutation likely occurring independently multiple times.

Curbing cancer’s lethality

Given AML’s near-limitless capacity for creating novel variants, what can clinicians do to halt the disease’s advance? According to Dr Maley, one approach would be to use cancer’s ability to evolve to our advantage, rather than attempt to fight it head on.

This paradigm draws on a branch of ecology known as life history theory. The idea is to carefully study the environmental factors that may lead organisms to favor either a fast or slow reproducing strategy to maximize their ability to survive.

According to the theory, fast reproduction tends to occur in environments with high extrinsic mortality. Aggressive cancer treatment creates just such an environment, favoring those cells able to reproduce quickly, producing large numbers of daughter cells, with a few evading extrinsic mortality to repopulate the tumor.

On the other hand, a very stable environment often favors slow reproduction, because organisms reach a carrying capacity of their surrounding environment. In this case, the limiting factor becomes competition between like organisms. Here, a slow reproducing strategy favoring greater investment in maintenance and survivability wins the competition.

“This approach would say, ‘Let’s keep tumors as stable as possible and keep their resources limited,’” Dr Maley said. “If we are able to keep the tumor cells contained and let them fight it out, we would expect to see more competitively fit cells that are growing very slowly.”

Total Therapy III, a combination treatment regimen of chemotherapy and radiation, is now commonly administered to patients with acute lymphoblastic leukemia. But this was not always the case.

In an interview with National Public Radio, childhood ALL patients James Eversull and Pat Patchell, now among the oldest of St. Jude Children’s Research Hospital’s leukemia survivors, reflect on their experiences with the once-experimental treatment.

Read and listen to the full interview at npr.org.

Total Therapy III, a combination treatment regimen of chemotherapy and radiation, is now commonly administered to patients with acute lymphoblastic leukemia. But this was not always the case.

In an interview with National Public Radio, childhood ALL patients James Eversull and Pat Patchell, now among the oldest of St. Jude Children’s Research Hospital’s leukemia survivors, reflect on their experiences with the once-experimental treatment.

Read and listen to the full interview at npr.org.

Total Therapy III, a combination treatment regimen of chemotherapy and radiation, is now commonly administered to patients with acute lymphoblastic leukemia. But this was not always the case.

In an interview with National Public Radio, childhood ALL patients James Eversull and Pat Patchell, now among the oldest of St. Jude Children’s Research Hospital’s leukemia survivors, reflect on their experiences with the once-experimental treatment.

Read and listen to the full interview at npr.org.

Lab mouse

In developing a compound that exhibits activity against a type of acute myeloid leukemia (AML), investigators have shown that transcription factors are, in fact, “druggable.”

The compound, AI-10-49, selectively binds to the mutated transcription factor CBFβ-SMMHC, and this prevents CBFβ-SMMHC from binding to another transcription factor, RUNX1.

In this way, AI-10-49 restores RUNX1 transcriptional activity, which leads to the death of inv(16) AML cells in vitro and in vivo.

“This drug that we’ve developed is . . . targeting a class of proteins that hasn’t been targeted for drug development very much in the past,” said study author John H. Bushweller, PhD, of the University of Virginia Health System in Charlottesville.

“It’s really a new paradigm, a new approach to try to treat these diseases. This class of proteins is very important for determining how much of many other proteins are made, so it’s a unique way of changing the way the cell behaves.”

Dr Bushweller and his colleagues described their work in Science.

The investigators noted that CBFβ-SMMHC is expressed in AML with the chromosome inversion inv(16)(p13q22). And CBFβ-SMMHC outcompetes wild-type CBFβ for binding to RUNX1, thereby deregulating RUNX1 activity in hematopoiesis and inducing AML.

“When you target a mutated protein in a cancer, you would ideally like to inhibit that mutated form of the protein but not affect the normal form of the protein that’s still there,” Dr Bushweller said. “In the case of [AI-10-49], we’ve achieved that.”

He and his colleagues tested AI-10-49 in 11 leukemia cells lines and found that only ME-1 cells were highly sensitive to the treatment. AI-10-49 effectively dissociated RUNX1 from CBFβ-SMMHC in ME-1 cells, with 90% dissociation after 6 hours of treatment.

But the drug had a modest effect on CBFβ-RUNX1 association. It also had negligible activity in normal human bone marrow cells.

The investigators then tested AI-10-49 in a mouse model of inv(16) AML. Control mice (vehicle-treated) succumbed to leukemia in a median of 33.5 days, compared to a median of 61 days for mice that received AI-10-49.

Dr Bushweller and his colleagues did not evaluate toxicity after long-term exposure to AI-10-49, but they found no evidence of toxicity after 7 days of AI-10-49 treatment.

Next, the investigators tested AI-10-49 in 4 primary inv(16) AML cell samples. They saw a reduction in leukemia cell viability when AI-10-49 was administered at 5 μM and 10 μM concentrations. Bivalent AI-10-49 was more potent than monovalent AI-10-47.

The team said these results suggest that direct inhibition of CBFβ-SMMHC may be an effective therapeutic approach for inv(16) AML, and the findings provide support for therapies targeting transcription factors.

Lab mouse

In developing a compound that exhibits activity against a type of acute myeloid leukemia (AML), investigators have shown that transcription factors are, in fact, “druggable.”

The compound, AI-10-49, selectively binds to the mutated transcription factor CBFβ-SMMHC, and this prevents CBFβ-SMMHC from binding to another transcription factor, RUNX1.

In this way, AI-10-49 restores RUNX1 transcriptional activity, which leads to the death of inv(16) AML cells in vitro and in vivo.

“This drug that we’ve developed is . . . targeting a class of proteins that hasn’t been targeted for drug development very much in the past,” said study author John H. Bushweller, PhD, of the University of Virginia Health System in Charlottesville.

“It’s really a new paradigm, a new approach to try to treat these diseases. This class of proteins is very important for determining how much of many other proteins are made, so it’s a unique way of changing the way the cell behaves.”

Dr Bushweller and his colleagues described their work in Science.

The investigators noted that CBFβ-SMMHC is expressed in AML with the chromosome inversion inv(16)(p13q22). And CBFβ-SMMHC outcompetes wild-type CBFβ for binding to RUNX1, thereby deregulating RUNX1 activity in hematopoiesis and inducing AML.

“When you target a mutated protein in a cancer, you would ideally like to inhibit that mutated form of the protein but not affect the normal form of the protein that’s still there,” Dr Bushweller said. “In the case of [AI-10-49], we’ve achieved that.”

He and his colleagues tested AI-10-49 in 11 leukemia cells lines and found that only ME-1 cells were highly sensitive to the treatment. AI-10-49 effectively dissociated RUNX1 from CBFβ-SMMHC in ME-1 cells, with 90% dissociation after 6 hours of treatment.

But the drug had a modest effect on CBFβ-RUNX1 association. It also had negligible activity in normal human bone marrow cells.

The investigators then tested AI-10-49 in a mouse model of inv(16) AML. Control mice (vehicle-treated) succumbed to leukemia in a median of 33.5 days, compared to a median of 61 days for mice that received AI-10-49.

Dr Bushweller and his colleagues did not evaluate toxicity after long-term exposure to AI-10-49, but they found no evidence of toxicity after 7 days of AI-10-49 treatment.

Next, the investigators tested AI-10-49 in 4 primary inv(16) AML cell samples. They saw a reduction in leukemia cell viability when AI-10-49 was administered at 5 μM and 10 μM concentrations. Bivalent AI-10-49 was more potent than monovalent AI-10-47.

The team said these results suggest that direct inhibition of CBFβ-SMMHC may be an effective therapeutic approach for inv(16) AML, and the findings provide support for therapies targeting transcription factors.

Lab mouse

In developing a compound that exhibits activity against a type of acute myeloid leukemia (AML), investigators have shown that transcription factors are, in fact, “druggable.”

The compound, AI-10-49, selectively binds to the mutated transcription factor CBFβ-SMMHC, and this prevents CBFβ-SMMHC from binding to another transcription factor, RUNX1.

In this way, AI-10-49 restores RUNX1 transcriptional activity, which leads to the death of inv(16) AML cells in vitro and in vivo.

“This drug that we’ve developed is . . . targeting a class of proteins that hasn’t been targeted for drug development very much in the past,” said study author John H. Bushweller, PhD, of the University of Virginia Health System in Charlottesville.

“It’s really a new paradigm, a new approach to try to treat these diseases. This class of proteins is very important for determining how much of many other proteins are made, so it’s a unique way of changing the way the cell behaves.”

Dr Bushweller and his colleagues described their work in Science.

The investigators noted that CBFβ-SMMHC is expressed in AML with the chromosome inversion inv(16)(p13q22). And CBFβ-SMMHC outcompetes wild-type CBFβ for binding to RUNX1, thereby deregulating RUNX1 activity in hematopoiesis and inducing AML.

“When you target a mutated protein in a cancer, you would ideally like to inhibit that mutated form of the protein but not affect the normal form of the protein that’s still there,” Dr Bushweller said. “In the case of [AI-10-49], we’ve achieved that.”

He and his colleagues tested AI-10-49 in 11 leukemia cells lines and found that only ME-1 cells were highly sensitive to the treatment. AI-10-49 effectively dissociated RUNX1 from CBFβ-SMMHC in ME-1 cells, with 90% dissociation after 6 hours of treatment.

But the drug had a modest effect on CBFβ-RUNX1 association. It also had negligible activity in normal human bone marrow cells.

The investigators then tested AI-10-49 in a mouse model of inv(16) AML. Control mice (vehicle-treated) succumbed to leukemia in a median of 33.5 days, compared to a median of 61 days for mice that received AI-10-49.

Dr Bushweller and his colleagues did not evaluate toxicity after long-term exposure to AI-10-49, but they found no evidence of toxicity after 7 days of AI-10-49 treatment.

Next, the investigators tested AI-10-49 in 4 primary inv(16) AML cell samples. They saw a reduction in leukemia cell viability when AI-10-49 was administered at 5 μM and 10 μM concentrations. Bivalent AI-10-49 was more potent than monovalent AI-10-47.

The team said these results suggest that direct inhibition of CBFβ-SMMHC may be an effective therapeutic approach for inv(16) AML, and the findings provide support for therapies targeting transcription factors.

The US Food and Drug Administration (FDA) has approved the leukocyte growth factor Zarxio (filgrastim-sndz), the first biosimilar product to be approved in the US.

A biosimilar product is approved based on data showing that it is highly similar to an already-approved biological product.

Sandoz Inc’s Zarxio is biosimilar to Amgen Inc’s Neupogen (filgrastim), which was originally licensed in 1991. Zarxio is now approved for the same indications as Neupogen.

Zarxio can be prescribed for:

• patients with cancer receiving myelosuppressive chemotherapy
• patients with acute myeloid leukemia receiving induction or consolidation chemotherapy
• patients with cancer undergoing bone marrow transplant
• patients undergoing autologous peripheral blood progenitor cell collection and therapy
• patients with severe chronic neutropenia.

Zarxio is marketed as Zarzio outside the US. The biosimilar is available in more than 60 countries worldwide.

“Biosimilars will provide access to important therapies for patients who need them,” said FDA Commissioner Margaret A. Hamburg, MD.

“Patients and the healthcare community can be confident that biosimilar products approved by the FDA meet the agency’s rigorous safety, efficacy, and quality standards.”

Zarxio data

The FDA’s approval of Zarxio is based on a review of evidence that included structural and functional characterization, in vivo data, human pharmacokinetic and pharmacodynamics data, clinical immunogenicity data, and other clinical safety and effectiveness data that demonstrates Zarxio is biosimilar to Neupogen.

The PIONEER study was the final piece of data the FDA used to approve Zarxio as biosimilar to Neupogen. The data was sufficient to allow extrapolation of the use of Zarxio to all indications of Neupogen.

In the PIONEER study, Zarxio and Neupogen both produced the expected reduction in the duration of severe neutropenia in cancer patients undergoing myelosuppressive chemotherapy—1.17 and 1.20 days, respectively.

The mean time to absolute neutrophil count recovery in cycle 1 was also similar—1.8 ± 0.97 days in the Zarxio arm and 1.7 ± 0.81 days in the Neupogen arm. No immunogenicity or antibodies against rhG-CSF were detected throughout the study.

The most common side effects of Zarxio are aching in the bones or muscles and redness, swelling, or itching at the injection site. Serious side effects may include spleen rupture; serious allergic reactions that may cause rash, shortness of breath, wheezing and/or swelling around the mouth and eyes; fast pulse and sweating; and acute respiratory distress syndrome.

About biosimilar approval

The Biologics Price Competition and Innovation Act of 2009 (BPCI Act) was passed as part of the Affordable Care Act that President Barack Obama signed into law in March 2010. The BPCI Act created an abbreviated licensure pathway for biological products shown to be “biosimilar” to or “interchangeable” with an FDA-licensed biological product, known as the reference product.

This abbreviated licensure pathway under section 351(k) of the Public Health Service Act permits reliance on certain existing scientific knowledge about the safety and effectiveness of the reference product, and it enables a biosimilar biological product to be licensed based on less than a full complement of product-specific preclinical and clinical data.

A biosimilar product can only be approved by the FDA if it has the same mechanism(s) of action, route(s) of administration, dosage form(s) and strength(s) as the reference product, and only for the indication(s) and condition(s) of use that have been approved for the reference product. The facilities where biosimilars are manufactured must also meet the FDA’s standards.

There must be no clinically meaningful differences between the biosimilar and the reference product in terms of safety and effectiveness. Only minor differences in clinically inactive components are allowable.

Zarxio has been approved as a biosimilar, not an interchangeable product. Under the BPCI Act, a biological product that has been approved as “interchangeable” may be substituted for the reference product without the intervention of the healthcare provider who prescribed the reference product.

For Zarxio’s approval, the FDA has designated a placeholder nonproprietary name for this product as “filgrastim-sndz.” The provision of a placeholder nonproprietary name should not be viewed as reflective of the agency’s decision on a comprehensive naming policy for biosimilars and other biological products.

While the FDA has not yet issued draft guidance on how current and future biological products marketed in the US should be named, the agency intends to do so in the near future.

For more details on Zarxio, see the full prescribing information.

The US Food and Drug Administration (FDA) has approved the leukocyte growth factor Zarxio (filgrastim-sndz), the first biosimilar product to be approved in the US.

A biosimilar product is approved based on data showing that it is highly similar to an already-approved biological product.

Sandoz Inc’s Zarxio is biosimilar to Amgen Inc’s Neupogen (filgrastim), which was originally licensed in 1991. Zarxio is now approved for the same indications as Neupogen.

Zarxio can be prescribed for:

• patients with cancer receiving myelosuppressive chemotherapy
• patients with acute myeloid leukemia receiving induction or consolidation chemotherapy
• patients with cancer undergoing bone marrow transplant
• patients undergoing autologous peripheral blood progenitor cell collection and therapy
• patients with severe chronic neutropenia.

Zarxio is marketed as Zarzio outside the US. The biosimilar is available in more than 60 countries worldwide.

“Biosimilars will provide access to important therapies for patients who need them,” said FDA Commissioner Margaret A. Hamburg, MD.

“Patients and the healthcare community can be confident that biosimilar products approved by the FDA meet the agency’s rigorous safety, efficacy, and quality standards.”

Zarxio data

The FDA’s approval of Zarxio is based on a review of evidence that included structural and functional characterization, in vivo data, human pharmacokinetic and pharmacodynamics data, clinical immunogenicity data, and other clinical safety and effectiveness data that demonstrates Zarxio is biosimilar to Neupogen.

The PIONEER study was the final piece of data the FDA used to approve Zarxio as biosimilar to Neupogen. The data was sufficient to allow extrapolation of the use of Zarxio to all indications of Neupogen.

In the PIONEER study, Zarxio and Neupogen both produced the expected reduction in the duration of severe neutropenia in cancer patients undergoing myelosuppressive chemotherapy—1.17 and 1.20 days, respectively.

The mean time to absolute neutrophil count recovery in cycle 1 was also similar—1.8 ± 0.97 days in the Zarxio arm and 1.7 ± 0.81 days in the Neupogen arm. No immunogenicity or antibodies against rhG-CSF were detected throughout the study.

The most common side effects of Zarxio are aching in the bones or muscles and redness, swelling, or itching at the injection site. Serious side effects may include spleen rupture; serious allergic reactions that may cause rash, shortness of breath, wheezing and/or swelling around the mouth and eyes; fast pulse and sweating; and acute respiratory distress syndrome.

About biosimilar approval

The Biologics Price Competition and Innovation Act of 2009 (BPCI Act) was passed as part of the Affordable Care Act that President Barack Obama signed into law in March 2010. The BPCI Act created an abbreviated licensure pathway for biological products shown to be “biosimilar” to or “interchangeable” with an FDA-licensed biological product, known as the reference product.

This abbreviated licensure pathway under section 351(k) of the Public Health Service Act permits reliance on certain existing scientific knowledge about the safety and effectiveness of the reference product, and it enables a biosimilar biological product to be licensed based on less than a full complement of product-specific preclinical and clinical data.

A biosimilar product can only be approved by the FDA if it has the same mechanism(s) of action, route(s) of administration, dosage form(s) and strength(s) as the reference product, and only for the indication(s) and condition(s) of use that have been approved for the reference product. The facilities where biosimilars are manufactured must also meet the FDA’s standards.

There must be no clinically meaningful differences between the biosimilar and the reference product in terms of safety and effectiveness. Only minor differences in clinically inactive components are allowable.

Zarxio has been approved as a biosimilar, not an interchangeable product. Under the BPCI Act, a biological product that has been approved as “interchangeable” may be substituted for the reference product without the intervention of the healthcare provider who prescribed the reference product.

For Zarxio’s approval, the FDA has designated a placeholder nonproprietary name for this product as “filgrastim-sndz.” The provision of a placeholder nonproprietary name should not be viewed as reflective of the agency’s decision on a comprehensive naming policy for biosimilars and other biological products.

While the FDA has not yet issued draft guidance on how current and future biological products marketed in the US should be named, the agency intends to do so in the near future.

For more details on Zarxio, see the full prescribing information.

The US Food and Drug Administration (FDA) has approved the leukocyte growth factor Zarxio (filgrastim-sndz), the first biosimilar product to be approved in the US.

A biosimilar product is approved based on data showing that it is highly similar to an already-approved biological product.

Sandoz Inc’s Zarxio is biosimilar to Amgen Inc’s Neupogen (filgrastim), which was originally licensed in 1991. Zarxio is now approved for the same indications as Neupogen.

Zarxio can be prescribed for:

• patients with cancer receiving myelosuppressive chemotherapy
• patients with acute myeloid leukemia receiving induction or consolidation chemotherapy
• patients with cancer undergoing bone marrow transplant
• patients undergoing autologous peripheral blood progenitor cell collection and therapy
• patients with severe chronic neutropenia.

Zarxio is marketed as Zarzio outside the US. The biosimilar is available in more than 60 countries worldwide.

“Biosimilars will provide access to important therapies for patients who need them,” said FDA Commissioner Margaret A. Hamburg, MD.

“Patients and the healthcare community can be confident that biosimilar products approved by the FDA meet the agency’s rigorous safety, efficacy, and quality standards.”

Zarxio data

The FDA’s approval of Zarxio is based on a review of evidence that included structural and functional characterization, in vivo data, human pharmacokinetic and pharmacodynamics data, clinical immunogenicity data, and other clinical safety and effectiveness data that demonstrates Zarxio is biosimilar to Neupogen.

The PIONEER study was the final piece of data the FDA used to approve Zarxio as biosimilar to Neupogen. The data was sufficient to allow extrapolation of the use of Zarxio to all indications of Neupogen.

In the PIONEER study, Zarxio and Neupogen both produced the expected reduction in the duration of severe neutropenia in cancer patients undergoing myelosuppressive chemotherapy—1.17 and 1.20 days, respectively.

The mean time to absolute neutrophil count recovery in cycle 1 was also similar—1.8 ± 0.97 days in the Zarxio arm and 1.7 ± 0.81 days in the Neupogen arm. No immunogenicity or antibodies against rhG-CSF were detected throughout the study.

The most common side effects of Zarxio are aching in the bones or muscles and redness, swelling, or itching at the injection site. Serious side effects may include spleen rupture; serious allergic reactions that may cause rash, shortness of breath, wheezing and/or swelling around the mouth and eyes; fast pulse and sweating; and acute respiratory distress syndrome.

About biosimilar approval

The Biologics Price Competition and Innovation Act of 2009 (BPCI Act) was passed as part of the Affordable Care Act that President Barack Obama signed into law in March 2010. The BPCI Act created an abbreviated licensure pathway for biological products shown to be “biosimilar” to or “interchangeable” with an FDA-licensed biological product, known as the reference product.

This abbreviated licensure pathway under section 351(k) of the Public Health Service Act permits reliance on certain existing scientific knowledge about the safety and effectiveness of the reference product, and it enables a biosimilar biological product to be licensed based on less than a full complement of product-specific preclinical and clinical data.

A biosimilar product can only be approved by the FDA if it has the same mechanism(s) of action, route(s) of administration, dosage form(s) and strength(s) as the reference product, and only for the indication(s) and condition(s) of use that have been approved for the reference product. The facilities where biosimilars are manufactured must also meet the FDA’s standards.

There must be no clinically meaningful differences between the biosimilar and the reference product in terms of safety and effectiveness. Only minor differences in clinically inactive components are allowable.

Zarxio has been approved as a biosimilar, not an interchangeable product. Under the BPCI Act, a biological product that has been approved as “interchangeable” may be substituted for the reference product without the intervention of the healthcare provider who prescribed the reference product.

For Zarxio’s approval, the FDA has designated a placeholder nonproprietary name for this product as “filgrastim-sndz.” The provision of a placeholder nonproprietary name should not be viewed as reflective of the agency’s decision on a comprehensive naming policy for biosimilars and other biological products.

While the FDA has not yet issued draft guidance on how current and future biological products marketed in the US should be named, the agency intends to do so in the near future.

For more details on Zarxio, see the full prescribing information.

For patients with secondary acute myeloid leukemia, induction therapy with cytarabine in combination with amonafide L-malate did not improve complete remission rates over the standard treatment of cytarabine and daunorubicin, investigators reported online March 2 in the Journal of Clinical Oncology.

Between January 2008 and August 2010, 433 patients with previously untreated secondary AML were randomly assigned to receive an intravenous infusion of cytarabine once per day for 7 days, plus either amonafide or daunorubicin for 4 days. The complete remission rate was 46% in the amonafide-plus-cytarabine group and 45% in the daunorubicin-plus-cytarabine group (P = .81), reported Dr. Richard M. Stone of Dana-Farber Cancer Institute, Boston, and associates.

“Although amonafide does not seem to provide benefit over standard induction therapy with daunorubicin, analyses of the data from this clinical trial may provide critical background information for tests of subsequent drugs that may have promise in this important disease subgroup, such as the liposomal-encapsulated daunorubicin/cytarabine CPX-351, the nuclear export inhibitor KPT-330, or the BCL-2 antagonist ABT-199,” they concluded.

Read the complete article here: (doi:10.1200/JCO.2014.57.0952).

For patients with secondary acute myeloid leukemia, induction therapy with cytarabine in combination with amonafide L-malate did not improve complete remission rates over the standard treatment of cytarabine and daunorubicin, investigators reported online March 2 in the Journal of Clinical Oncology.

Between January 2008 and August 2010, 433 patients with previously untreated secondary AML were randomly assigned to receive an intravenous infusion of cytarabine once per day for 7 days, plus either amonafide or daunorubicin for 4 days. The complete remission rate was 46% in the amonafide-plus-cytarabine group and 45% in the daunorubicin-plus-cytarabine group (P = .81), reported Dr. Richard M. Stone of Dana-Farber Cancer Institute, Boston, and associates.

“Although amonafide does not seem to provide benefit over standard induction therapy with daunorubicin, analyses of the data from this clinical trial may provide critical background information for tests of subsequent drugs that may have promise in this important disease subgroup, such as the liposomal-encapsulated daunorubicin/cytarabine CPX-351, the nuclear export inhibitor KPT-330, or the BCL-2 antagonist ABT-199,” they concluded.

Read the complete article here: (doi:10.1200/JCO.2014.57.0952).

For patients with secondary acute myeloid leukemia, induction therapy with cytarabine in combination with amonafide L-malate did not improve complete remission rates over the standard treatment of cytarabine and daunorubicin, investigators reported online March 2 in the Journal of Clinical Oncology.

Between January 2008 and August 2010, 433 patients with previously untreated secondary AML were randomly assigned to receive an intravenous infusion of cytarabine once per day for 7 days, plus either amonafide or daunorubicin for 4 days. The complete remission rate was 46% in the amonafide-plus-cytarabine group and 45% in the daunorubicin-plus-cytarabine group (P = .81), reported Dr. Richard M. Stone of Dana-Farber Cancer Institute, Boston, and associates.

“Although amonafide does not seem to provide benefit over standard induction therapy with daunorubicin, analyses of the data from this clinical trial may provide critical background information for tests of subsequent drugs that may have promise in this important disease subgroup, such as the liposomal-encapsulated daunorubicin/cytarabine CPX-351, the nuclear export inhibitor KPT-330, or the BCL-2 antagonist ABT-199,” they concluded.

Read the complete article here: (doi:10.1200/JCO.2014.57.0952).

Saad S. Kenderian

Photo courtesy of

BMT Tandem Meetings

SAN DIEGO—Researchers have developed a “biodegradable” chimeric antigen receptor (CAR) T-cell therapy that could potentially serve as a preparative regimen for acute myeloid leukemia (AML) patients undergoing allogeneic transplant.

The team created CAR T cells that target CD33 (CART33) and modified them with RNA so the cells would stop expressing CARs over time.

In mouse models of AML, the RNA-CART33 cells had an antileukemic effect and induced myeloablation.

The cells also stopped expressing CARs by the 2-week mark, which would allow for engraftment after allogeneic transplant, according to the researchers.

Saad S. Kenderian, MD, of the University of Pennsylvania in Philadelphia, presented this research at the 2015 BMT Tandem Meetings as one of the meeting’s “Best Abstracts” (abstract 1). The research was funded by Novartis.

“Allogeneic transplantation is the only potentially curative option in relapsed/refractory AML,” Dr Kenderian noted. “Outcomes are poor if patients are transplanted in residual disease  . . . , and these patients are often considered transplant-ineligible. Therefore, novel therapies are desperately needed.”

With this in mind, Dr Kenderian and his colleagues set out to develop a CAR T-cell therapy targeting CD33, which is expressed on AML blasts.

The researchers created a CAR from the anti-CD33 single-chain fragment variable of gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. They transduced T cells with this construct and expanded them in culture using anti-CD3/CD28 magnetic beads.

The team then tested these CART33 cells in NSGS mice engrafted with primary AML blasts. The mice received CART33 cells, another CAR T-cell therapy known as CART123, or control T cells.

At 4 weeks, mice that had received CART33 or CART123 cells were entirely leukemia-free, but the disease continued to progress in mice that received control T cells.

Likewise, when the experiment ended at 200 days, survival was 100% among mice that received CART33 or CART123, but all of the control mice had died. And at 200 days, CAR T cells were still circulating in the CART33- and CART123-treated mice.

Next, the researchers administered CART33 cells to HIS-NSG mice engrafted with human bone marrow and found the treatment resulted in myeloablation. There was a significant reduction of CD34-positive cells in mice that received CART33 compared to mice that received control T cells or no treatment.

“So based on our preclinical data, when we treat refractory AML with lentivirally transduced CART33, that will result in myeloablation, eradication of AML, and persistence of these CARs,” Dr Kenderian said.

“If allogeneic transplantation is performed at this aplastic stage, it will likely lead to rejection of the graft by persisting CAR therapy, which also means that elimination of CARs is necessary prior to stem cell infusion.”

So the researchers decided to create a transiently expressed, mRNA-modified CAR based on CART33. They electroporated T cells with this construct, and the cells expressed CARs for up to 6 days.

In experiments with the MOLM14 cell line, RNA-modified CART33 cells exhibited transient but comparable killing ability as LV-transduced CART33.

The researchers then tested RNA-CART33 in combination with chemotherapy in vivo. They transplanted NSG mice with MOLM14 and treated them with cyclophosphamide plus RNA-CART33 or cyclophosphamide plus control T cells.

Combination RNA-CART33 and chemotherapy prompted stronger, more durable antileukemic activity than cyclophosphamide and control T cells. Furthermore, there was a significant improvement in survival among RNA-CART33-treated mice (P=0.01).

Finally, Dr Kenderian and his colleagues tested the effect of RNA-CART33 on hematopoiesis. The team treated NSGS mice with busulfan and transplanted them with T-cell-depleted bone marrow. Following engraftment, mice received RNA-CART33 cells, LV-CART33 cells, or control T cells.

The researchers followed the mice for 2 weeks and found that both RNA-CART33 and LV-CART33 induced myeloablation. And at 14 days, LV-CART33-treated mice were still expressing CARs, but RNA-CART33-treated mice were not.

“Based on our preclinical data, if we treat refractory AML with RNA-modified CART33, that results in myeloablation, anti-AML activity, and biodegradable, non-persisting CARs,” Dr Kenderian summarized.

“If allogeneic transplantation follows at this stage, it will likely lead to engraftment. Therefore, we conclude from this study that RNA-CART33 could be incorporated in novel conditioning regimens and will be tested in pilot phase 1 studies.”

Saad S. Kenderian

Photo courtesy of

BMT Tandem Meetings

SAN DIEGO—Researchers have developed a “biodegradable” chimeric antigen receptor (CAR) T-cell therapy that could potentially serve as a preparative regimen for acute myeloid leukemia (AML) patients undergoing allogeneic transplant.

The team created CAR T cells that target CD33 (CART33) and modified them with RNA so the cells would stop expressing CARs over time.

In mouse models of AML, the RNA-CART33 cells had an antileukemic effect and induced myeloablation.

The cells also stopped expressing CARs by the 2-week mark, which would allow for engraftment after allogeneic transplant, according to the researchers.

Saad S. Kenderian, MD, of the University of Pennsylvania in Philadelphia, presented this research at the 2015 BMT Tandem Meetings as one of the meeting’s “Best Abstracts” (abstract 1). The research was funded by Novartis.

“Allogeneic transplantation is the only potentially curative option in relapsed/refractory AML,” Dr Kenderian noted. “Outcomes are poor if patients are transplanted in residual disease  . . . , and these patients are often considered transplant-ineligible. Therefore, novel therapies are desperately needed.”

With this in mind, Dr Kenderian and his colleagues set out to develop a CAR T-cell therapy targeting CD33, which is expressed on AML blasts.

The researchers created a CAR from the anti-CD33 single-chain fragment variable of gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. They transduced T cells with this construct and expanded them in culture using anti-CD3/CD28 magnetic beads.

The team then tested these CART33 cells in NSGS mice engrafted with primary AML blasts. The mice received CART33 cells, another CAR T-cell therapy known as CART123, or control T cells.

At 4 weeks, mice that had received CART33 or CART123 cells were entirely leukemia-free, but the disease continued to progress in mice that received control T cells.

Likewise, when the experiment ended at 200 days, survival was 100% among mice that received CART33 or CART123, but all of the control mice had died. And at 200 days, CAR T cells were still circulating in the CART33- and CART123-treated mice.

Next, the researchers administered CART33 cells to HIS-NSG mice engrafted with human bone marrow and found the treatment resulted in myeloablation. There was a significant reduction of CD34-positive cells in mice that received CART33 compared to mice that received control T cells or no treatment.

“So based on our preclinical data, when we treat refractory AML with lentivirally transduced CART33, that will result in myeloablation, eradication of AML, and persistence of these CARs,” Dr Kenderian said.

“If allogeneic transplantation is performed at this aplastic stage, it will likely lead to rejection of the graft by persisting CAR therapy, which also means that elimination of CARs is necessary prior to stem cell infusion.”

So the researchers decided to create a transiently expressed, mRNA-modified CAR based on CART33. They electroporated T cells with this construct, and the cells expressed CARs for up to 6 days.

In experiments with the MOLM14 cell line, RNA-modified CART33 cells exhibited transient but comparable killing ability as LV-transduced CART33.

The researchers then tested RNA-CART33 in combination with chemotherapy in vivo. They transplanted NSG mice with MOLM14 and treated them with cyclophosphamide plus RNA-CART33 or cyclophosphamide plus control T cells.

Combination RNA-CART33 and chemotherapy prompted stronger, more durable antileukemic activity than cyclophosphamide and control T cells. Furthermore, there was a significant improvement in survival among RNA-CART33-treated mice (P=0.01).

Finally, Dr Kenderian and his colleagues tested the effect of RNA-CART33 on hematopoiesis. The team treated NSGS mice with busulfan and transplanted them with T-cell-depleted bone marrow. Following engraftment, mice received RNA-CART33 cells, LV-CART33 cells, or control T cells.

The researchers followed the mice for 2 weeks and found that both RNA-CART33 and LV-CART33 induced myeloablation. And at 14 days, LV-CART33-treated mice were still expressing CARs, but RNA-CART33-treated mice were not.

“Based on our preclinical data, if we treat refractory AML with RNA-modified CART33, that results in myeloablation, anti-AML activity, and biodegradable, non-persisting CARs,” Dr Kenderian summarized.

“If allogeneic transplantation follows at this stage, it will likely lead to engraftment. Therefore, we conclude from this study that RNA-CART33 could be incorporated in novel conditioning regimens and will be tested in pilot phase 1 studies.”

Saad S. Kenderian

Photo courtesy of

BMT Tandem Meetings

SAN DIEGO—Researchers have developed a “biodegradable” chimeric antigen receptor (CAR) T-cell therapy that could potentially serve as a preparative regimen for acute myeloid leukemia (AML) patients undergoing allogeneic transplant.

The team created CAR T cells that target CD33 (CART33) and modified them with RNA so the cells would stop expressing CARs over time.

In mouse models of AML, the RNA-CART33 cells had an antileukemic effect and induced myeloablation.

The cells also stopped expressing CARs by the 2-week mark, which would allow for engraftment after allogeneic transplant, according to the researchers.

Saad S. Kenderian, MD, of the University of Pennsylvania in Philadelphia, presented this research at the 2015 BMT Tandem Meetings as one of the meeting’s “Best Abstracts” (abstract 1). The research was funded by Novartis.

“Allogeneic transplantation is the only potentially curative option in relapsed/refractory AML,” Dr Kenderian noted. “Outcomes are poor if patients are transplanted in residual disease  . . . , and these patients are often considered transplant-ineligible. Therefore, novel therapies are desperately needed.”

With this in mind, Dr Kenderian and his colleagues set out to develop a CAR T-cell therapy targeting CD33, which is expressed on AML blasts.

The researchers created a CAR from the anti-CD33 single-chain fragment variable of gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. They transduced T cells with this construct and expanded them in culture using anti-CD3/CD28 magnetic beads.

The team then tested these CART33 cells in NSGS mice engrafted with primary AML blasts. The mice received CART33 cells, another CAR T-cell therapy known as CART123, or control T cells.

At 4 weeks, mice that had received CART33 or CART123 cells were entirely leukemia-free, but the disease continued to progress in mice that received control T cells.

Likewise, when the experiment ended at 200 days, survival was 100% among mice that received CART33 or CART123, but all of the control mice had died. And at 200 days, CAR T cells were still circulating in the CART33- and CART123-treated mice.

Next, the researchers administered CART33 cells to HIS-NSG mice engrafted with human bone marrow and found the treatment resulted in myeloablation. There was a significant reduction of CD34-positive cells in mice that received CART33 compared to mice that received control T cells or no treatment.

“So based on our preclinical data, when we treat refractory AML with lentivirally transduced CART33, that will result in myeloablation, eradication of AML, and persistence of these CARs,” Dr Kenderian said.

“If allogeneic transplantation is performed at this aplastic stage, it will likely lead to rejection of the graft by persisting CAR therapy, which also means that elimination of CARs is necessary prior to stem cell infusion.”

So the researchers decided to create a transiently expressed, mRNA-modified CAR based on CART33. They electroporated T cells with this construct, and the cells expressed CARs for up to 6 days.

In experiments with the MOLM14 cell line, RNA-modified CART33 cells exhibited transient but comparable killing ability as LV-transduced CART33.

The researchers then tested RNA-CART33 in combination with chemotherapy in vivo. They transplanted NSG mice with MOLM14 and treated them with cyclophosphamide plus RNA-CART33 or cyclophosphamide plus control T cells.

Combination RNA-CART33 and chemotherapy prompted stronger, more durable antileukemic activity than cyclophosphamide and control T cells. Furthermore, there was a significant improvement in survival among RNA-CART33-treated mice (P=0.01).

Finally, Dr Kenderian and his colleagues tested the effect of RNA-CART33 on hematopoiesis. The team treated NSGS mice with busulfan and transplanted them with T-cell-depleted bone marrow. Following engraftment, mice received RNA-CART33 cells, LV-CART33 cells, or control T cells.

The researchers followed the mice for 2 weeks and found that both RNA-CART33 and LV-CART33 induced myeloablation. And at 14 days, LV-CART33-treated mice were still expressing CARs, but RNA-CART33-treated mice were not.

“Based on our preclinical data, if we treat refractory AML with RNA-modified CART33, that results in myeloablation, anti-AML activity, and biodegradable, non-persisting CARs,” Dr Kenderian summarized.

“If allogeneic transplantation follows at this stage, it will likely lead to engraftment. Therefore, we conclude from this study that RNA-CART33 could be incorporated in novel conditioning regimens and will be tested in pilot phase 1 studies.”