Atsushi Nonami

Requirement for CDK6 in MLL-rearranged acute myeloid leukemia

Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts.

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

Objectives Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment.

Identification of Wee1 as a novel therapeutic target for mutant RAS-driven acute leukemia and other malignancies.

Direct targeting of rat sarcoma (RAS), which is frequently mutated, has proven to be challenging, and inhibition of individual downstream RAS mediators has resulted in limited clinical efficacy. We designed a chemical screen to identify compounds capable of potentiating mammalian target of rapamycin (mTOR) inhibition in mutant RAS-positive leukemia, and identified a Wee1 inhibitor. Synergy was observed in both mutant neuroblastoma RAS viral oncogene homolog (NRAS)- and mutant kirsten RAS viral oncogene homolog (KRAS)-positive acute myelogenous leukemia (AML) cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-binding protein 1 and s6 kinase, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 knockdown, as well as partial reversal of drug combination-induced apoptosis by a cyclin-dependent kinase 1 (CDK1) inhibitor. Importantly, we also extended our findings to other mutant RAS-expressing malignancies, including mutant NRAS-positive melanoma, and mutant KRAS-positive colorectal cancer, pancreatic cancer and lung cancer. We observed favorable responses with combined Wee1/mTOR inhibition in human cancer cell lines from multiple malignancies, and inhibition of tumor growth in in vivo models of mutant KRAS lung cancer and leukemia. The present study introduces for the first time Wee1 inhibition combined with mTOR inhibition as a novel therapeutic strategy for the selective treatment of mutant RAS-positive leukemia and other mutant RAS-expressing malignancies.

Discovery of a BTK/MNK dual inhibitor for lymphoma and leukemia.

Bruton’s tyrosine kinase (BTK) kinase is a member of the TEC kinase family and is a key regulator of the B-cell receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Pharmacological inhibition of BTK is clinically effective against a variety of B-cell malignances, such as mantle cell lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and activated B-cell–diffuse large B-cell lymphoma. MNK kinase is one of the key downstream regulators in the RAF–MEK–ERK signaling pathway and controls protein synthesis via regulating the activity of eIF4E. Inhibition of MNK activity has been observed to moderately inhibit the proliferation of AML cells. Through a structure-based drug-design approach, we have discovered a selective and potent BTK/MNK dual kinase inhibitor (QL-X-138), which exhibits covalent binding to BTK and noncovalent binding to MNK. Compared with the BTK kinase inhibitor (PCI-32765) and the MNK kinase inhibitor (cercosporamide), QL-X-138 enhanced the antiproliferative efficacies in vitro against a variety of B-cell cancer cell lines, as well as AML and CLL primary patient cells, which respond moderately to BTK inhibitor in vitro. The agent can effectively arrest the growth of lymphoma and leukemia cells at the G0–G1 stage and can induce strong apoptotic cell death. These primary results demonstrate that simultaneous inhibition of BTK and MNK kinase activity might be a new therapeutic strategy for B-cell malignances.

Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.

Pathological glycogenesis through glycogen synthase 1 and suppression of excessive AMP kinase activity in myeloid leukemia cells

The rapid proliferation of myeloid leukemia cells is highly dependent on increased glucose metabolism. Through an unbiased metabolomics analysis of leukemia cells, we found that the glycogenic precursor UDP-D-glucose is pervasively upregulated, despite low glycogen levels. Targeting the rate-limiting glycogen synthase 1 (GYS1) not only decreased glycolytic flux but also increased activation of the glycogen-responsive AMP kinase (AMPK), leading to significant growth suppression. Further, genetic and pharmacological hyper-activation of AMPK was sufficient to induce the changes observed with GYS1 targeting. Cancer genomics data also indicate that elevated levels of the glycogenic enzymes GYS1/2 or GBE1 (glycogen branching enzyme 1) are associated with poor survival in AML. These results suggest a novel mechanism whereby leukemic cells sustain aberrant proliferation by suppressing excess AMPK activity through elevated glycogenic flux and provide a therapeutic entry point for targeting leukemia cell metabolism.

Ibrutinib selectively targets FLT3-ITD in mutant FLT3-positive AML.

Ibrutinib (PCI-32765) is an irreversible BTK (Bruton’s tyrosine kinase) kinase inhibitor that has been extensively used as a tool compound to validate the role of BTK kinase in B cell related malignances. Ibrutinib has been shown in preclinical studies to inhibit the proliferation of diffuse large B-cell lymphoma cells, mantle cell lymphoma cells, chronic lymphocytic leukemia cells and multiple myeloma cells by blocking BTK kinase activity; ibrutinib was recently approved for the clinical application on mantle cell lymphoma and chronic lymphocytic leukemia cells. Ibrutinib has also exhibited anti-inflammatory effects in preclinical models. Recently, it has been reported that ibrutinib is also effective against epidermal growth factor receptor mutantpositive non-small cell lung cancers through inhibition of epidermal growth factor receptor kinase activities. In addition, there is evidence showing that BTK is also an important target for Acute Myeloid Leukemia (AML). Despite the evidence that BTK knockdown impaired AML cancer cell growth, which suggested that BTK was important for AML cell proliferation, BTK kinase inhibition through use of a small molecule inhibitor like ibrutinib led only to moderate inhibition of proliferation of U937 cells with no apparent activity against other AML cell lines such as HL-60, TF-1 and THP-1. To further investigate the potency and activity of ibrutinib against AML, we screened a panel of AML cell lines spanning M0–M7 disease stages. Interestingly, we found that only FLT3-internal tandem duplication (ITD) mutant AML cell lines (MOLM13, MOLM14 and MV4-11) were sensitive to ibrutinib (Figure 1a and Supplementary Table 1). This is similar to what has been observed with the highly

Identification of novel therapeutic targets in acute leukemias with NRAS mutations using a pharmacologic approach

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.

Upregulation of IGF1R by Mutant RAS in Leukemia and Potentiation of RAS Signaling Inhibitors by Small-Molecule Inhibition of IGF1R

Purpose: Activating mutations in the RAS oncogene occur frequently in human leukemias. Direct targeting of RAS has proven to be challenging, although targeting of downstream RAS mediators, such as MEK, is currently being tested clinically. Given the complexity of RAS signaling, it is likely that combinations of targeted agents will be more effective than single agents. Experimental Design: A chemical screen using RAS-dependent leukemia cells was developed to identify compounds with unanticipated activity in the presence of an MEK inhibitor and led to identification of inhibitors of IGF1R. Results were validated using cell-based proliferation, apoptosis, cell-cycle, and gene knockdown assays; immunoprecipitation and immunoblotting; and a noninvasive in vivo bioluminescence model of acute myeloid leukemia (AML). Results: Mechanistically, IGF1R protein expression/activity was substantially increased in mutant RAS-expressing cells, and suppression of RAS led to decreases in IGF1R. Synergy between MEK and IGF1R inhibitors correlated with induction of apoptosis, inhibition of cell-cycle progression, and decreased phospho-S6 and phospho-4E-BP1. In vivo, NSG mice tail veins injected with OCI-AML3-luc+ cells showed significantly lower tumor burden following 1 week of daily oral administration of 50 mg/kg NVP-AEW541 (IGF1R inhibitor) combined with 25 mg/kg AZD6244 (MEK inhibitor), as compared with mice treated with either agent alone. Drug combination effects observed in cell-based assays were generalized to additional mutant RAS-positive neoplasms. Conclusions: The finding that downstream inhibitors of RAS signaling and IGF1R inhibitors have synergistic activity warrants further clinical investigation of IGF1R and RAS signaling inhibition as a potential treatment strategy for RAS-driven malignancies. Clin Cancer Res; 20(21); 5483–95. ©2014 AACR.

Inhibition of USP10 induces degradation of oncogenic FLT3

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.