Martin Carroll

The Common Feature of Leukemia-Associated IDH1 and IDH2 Mutations Is a Neomorphic Enzyme Activity Converting α-Ketoglutarate to 2-Hydroxyglutarate

The somatic mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) observed in gliomas can lead to the production of 2-hydroxyglutarate (2HG). Here, we report that tumor 2HG is elevated in a high percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Surprisingly, less than half of cases with elevated 2HG possessed IDH1 mutations. The remaining cases with elevated 2HG had mutations in IDH2, the mitochondrial homolog of IDH1. These data demonstrate that a shared feature of all cancer-associated IDH mutations is production of the oncometabolite 2HG. Furthermore, AML patients with IDH mutations display a significantly reduced number of other well characterized AML-associated mutations and/or associated chromosomal abnormalities, potentially implicating IDH mutation in a distinct mechanism of AML pathogenesis.

Distinct IL-2 Receptor Signaling Pattern in CD4+CD25 + Regulatory T Cells

Despite expression of the high-affinity IL-2R, CD4+CD25+ regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4+CD25+ T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4+CD25+ Tregs to IL-2. IL-2R stimulation results in a G1 cell cycle arrest, cellular enlargement and increased cellular survival of CD4+CD25+ T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4+CD25+ T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.

Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice

Human leukemic stem cells, like other cancer stem cells, are hypothesized to be rare, capable of incomplete differentiation, and restricted to a phenotype associated with early hematopoietic progenitors or stem cells. However, recent work in other types of tumors has challenged the cancer stem cell model. Using a robust model of xenotransplantation based on NOD/SCID/IL2Rγc-deficient mice, we confirmed that human leukemic stem cells, functionally defined by us as SCID leukemia-initiating cells (SL-ICs), are rare in acute myelogenous leukemia (AML). In contrast to previous results, SL-ICs were found among cells expressing lineage markers (i.e., among Lin+ cells), CD38, or CD45RA, all markers associated with normal committed progenitors. Remarkably, each engrafting fraction consistently recapitulated the original phenotypic diversity of the primary AML specimen and contained self-renewing leukemic stem cells, as demonstrated by secondary transplants. While SL-ICs were enriched in the Lin-CD38- fraction compared with the other fractions analyzed, SL-ICs in this fraction represented only one-third of all SL-ICs present in the unfractionated specimen. These results indicate that human AML stem cells are rare and enriched but not restricted to the phenotype associated with normal primitive hematopoietic cells. These results suggest a plasticity of the cancer stem cell phenotype that we believe has not been previously described.

AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3

AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3

Proteomic and genetic approaches identify Syk as an AML target.

Cell-based screening can facilitate the rapid identification of compounds inducing complex cellular phenotypes. Advancing a compound toward the clinic, however, generally requires the identification of precise mechanisms of action. We previously found that epidermal growth factor receptor (EGFR) inhibitors induce acute myeloid leukemia (AML) differentiation via a non-EGFR mechanism. In this report, we integrated proteomic and RNAi-based strategies to identify their off-target, anti-AML mechanism. These orthogonal approaches identified Syk as a target in AML. Genetic and pharmacological inactivation of Syk with a drug in clinical trial for other indications promoted differentiation of AML cells and attenuated leukemia growth in vivo. These results demonstrate the power of integrating diverse chemical, proteomic, and genomic screening approaches to identify therapeutic strategies for cancer.

Fatal myeloproliferation, induced in mice by TEL/PDGFbetaR expression, depends on PDGFbetaR tyrosines 579/581.

The t(5;12)(q33;p13) translocation associated with chronic myelomonocytic leukemia (CMML) generates a TEL/PDGFbetaR fusion gene. Here, we used a murine bone marrow transplant (BMT) assay to test the transforming properties of TEL/PDGFbetaR in vivo. TEL/PDGFbetaR, introduced into whole bone marrow by retroviral transduction, caused a rapidly fatal myeloproliferative disease that closely recapitulated human CMML. TEL/PDGFbetaR transplanted mice developed leukocytosis with Gr-1(+) granulocytes, splenomegaly, evidence of extramedullary hematopoiesis, and bone marrow fibrosis, but no lymphoproliferative disease. We assayed mutant forms of the TEL/PDGFbetaR fusion protein - including 8 tyrosine to phenylalanine substitutions at phosphorylated PDGFbetaR sites to which various SH2 domain-containing signaling intermediates bind - for ability to transform hematopoietic cells. All of the phenylalanine (F-) mutants tested conferred IL-3-independence to a cultured murine hematopoietic cell line, but, in the BMT assay, different F-mutants displayed distinct transforming properties. In transplanted animals, tyrosines 579/581 proved critical for the development of myeloproliferative phenotype. F-mutants with these residues mutated showed no sign of myeloproliferation but instead developed T-cell lymphomas. In summary, TEL/PDGFbetaR is necessary and sufficient to induce a myeloproliferative disease in a murine BMT model, and PDGFbetaR residues Y579/581 are required for this phenotype.

CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

BCR/ABL translocates to the nucleus and disrupts an ATR-dependent intra-S phase checkpoint.

Chronic myelogeneous leukemia (CML) is a two-stage disease associated with expression of the BCR/ABL tyrosine kinase protein. However, whether BCR/ABL expression directly causes blast crisis, and if so by what mechanism, is unknown. We have found that BCR/ABL translocates from the cytoplasm to the nucleus after genotoxic stress. Furthermore, BCR/ABL increases DNA double-strand damage after etoposide treatment and leads to a defect in an intra-S phase checkpoint, causing a radioresistant DNA synthesis (RDS) phenotype. In the nucleus, BCR/ABL associates with the ataxia-telangiectasia and rad 3-related protein (ATR) and disrupts ATR-dependent signal transduction. Overexpression of ATR in a BCR/ABL-expressing cell line corrects the DNA damage phenotype. These results demonstrate a nuclear role for BCR/ABL in altering the cellular response to DNA damage.

mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL.

DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies

The development of effective treatment strategies for most forms of acute myeloid leukemia (AML) has languished for the past several decades. There are a number of reasons for this, but key among them is the considerable heterogeneity of this disease and the paucity of molecular markers that can be used to predict clinical outcomes and responsiveness to different therapies. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatment that are based on individual mutational profiles. It is particularly notable that studies by The Cancer Genome Atlas and others have determined that 44% of patients with AML exhibit mutations in genes that regulate methylation of genomic DNA. In particular, frequent mutation has been observed in the genes encoding DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), as well as Tet oncogene family member 2. This review will summarize the incidence of these mutations, their impact on biochemical functions including epigenetic modification of genomic DNA and their potential usefulness as prognostic indicators. Importantly, the presence of DNMT3A, IDH1 or IDH2 mutations may confer sensitivity to novel therapeutic approaches, including the use of demethylating agents. Therefore, the clinical experience with decitabine and azacitidine in the treatment of patients harboring these mutations will be reviewed. Overall, we propose that understanding the role of these mutations in AML biology will lead to more rational therapeutic approaches targeting molecularly defined subtypes of the disease.