Matthew C. Stubbs

Transformation from committed progenitor to leukaemia stem cell initiated by MLL–AF9

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL–AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.

HOXA9 is required for survival in human MLL-rearranged acute leukemias.

Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes, including HOXA9. Here, we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias using RNA interference. Gene expression profiling after HOXA9 suppression demonstrated co-down-regulation of a program highly expressed in human MLL-AML and murine MLL-leukemia stem cells, including HOXA10, MEIS1, PBX3, and MEF2C. We demonstrate that HOXA9 depletion in 17 human AML/ALL cell lines (7 MLL-rearranged, 10 MLL-germline) induces proliferation arrest and apoptosis specifically in MLL-rearranged cells (P = .007). Similarly, assessment of primary AMLs demonstrated that HOXA9 suppression induces apoptosis to a greater extent in MLL-rearranged samples (P = .01). Moreover, mice transplanted with HOXA9-depleted t(4;11) SEMK2 cells revealed a significantly lower leukemia burden, thus identifying a role for HOXA9 in leukemia survival in vivo. Our data indicate an important role for HOXA9 in human MLL-rearranged leukemias and suggest that targeting HOXA9 or downstream programs may be a novel therapeutic option.

Cell of origin determines clinically relevant subtypes of MLL-rearranged AML.

Mixed lineage leukemia (MLL)-fusion proteins can induce acute myeloid leukemias (AMLs) from either hematopoietic stem cells (HSCs) or granulocyte–macrophage progenitors (GMPs), but it remains unclear whether the cell of origin influences the biology of the resultant leukemia. MLL-AF9-transduced single HSCs or GMPs could be continuously replated, but HSC-derived clones were more likely than GMP-derived clones to initiate AML in mice. Leukemia stem cells derived from either HSCs or GMPs had a similar immunophenotype consistent with a maturing myeloid cell (LGMP). Gene expression analyses demonstrated that LGMP inherited gene expression programs from the cell of origin including high-level Evi-1 expression in HSC-derived LGMP. The gene expression signature of LGMP derived from HSCs was enriched in poor prognosis human MLL-rearranged AML in three independent data sets. Moreover, global 5′-mC levels were elevated in HSC-derived leukemias as compared with GMP-derived leukemias. This mirrored a difference seen in 5′-mC between MLL-rearranged human leukemias that are either EVI1 positive or EVI1 negative. Finally, HSC-derived leukemias were more resistant to chemotherapy than GMP-derived leukemias. These data demonstrate that the cell of origin influences the gene expression profile, the epigenetic state and the drug response in AML, and that these differences can account for clinical heterogeneity within a molecularly defined group of leukemias.

MLL-AF9 and FLT3 cooperation in acute myelogenous leukemia: development of a model for rapid therapeutic assessment.

Human leukemias harboring chromosomal translocations involving the mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation. We demonstrate that MLL-AF9 expression induces acute myelogenous leukemia (AML) in approximately 70 days, whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. Secondary transplantation of splenic cells from diseased mice established that leukemia stem cells are present at a very high frequency of approximately 1:100 in both diseases. Importantly, prospectively isolated granulocyte macrophage progenitors (GMPs) coinfected with MLL-AF9 and FLT3-ITD give rise to a similar AML, with shorter latency than from GMP transduced with MLL-AF9 alone. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using noninvasive bioluminescence imaging. We used this model to demonstrate that MLL-AF9/FLT3-ITD-induced leukemias are sensitive to FLT3 inhibition in a 2–3 week in vivo assay. These data show that activated FLT3 cooperates with MLL-AF9 to accelerate onset of an AML from whole bone marrow as well as a committed hematopoietic progenitor, and provide a new genetically defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.

Therapeutic Implications of Leukemia Stem Cell Development

Acute myelogenous leukemias, and perhaps many other cancers, are maintained by a population of cancer stem cells that can regenerate themselves as well as give rise to more differentiated and less proliferative cells that constitute the bulk of the disease. Recent discoveries have shed light on both the nature of leukemia stem cells (LSC) and their cells of origin. Here, we review which hematopoietic cells could give rise to LSC, and the phenotype of fully developed LSC. The perturbed developmental pathways and cellular context of LSC development have implications for the development of new therapeutic approaches.

Selective Inhibition of HDAC1 and HDAC2 as a Potential Therapeutic Option for B-ALL

Purpose: Histone deacetylase inhibitors (HDACi) have recently emerged as efficacious therapies that target epigenetic mechanisms in hematologic malignancies. One such hematologic malignancy, B-cell acute lymphoblastic leukemia (B-ALL), may be highly dependent on epigenetic regulation for leukemia development and maintenance, and thus sensitive to small-molecule inhibitors that target epigenetic mechanisms. Experimental Design: A panel of B-ALL cell lines was tested for sensitivity to HDACi with varying isoform sensitivity. Isoform-specific shRNAs were used as further validation of HDACs as relevant therapeutic targets in B-ALL. Mouse xenografts of B-cell malignancy–derived cell lines and a pediatric B-ALL were used to demonstrate pharmacologic efficacy. Results: Nonselective HDAC inhibitors were cytotoxic to a panel of B-ALL cell lines as well as to xenografted human leukemia patient samples. Assessment of isoform-specific HDACi indicated that targeting HDAC1-3 with class I HDAC-specific inhibitors was sufficient to inhibit growth of B-ALL cell lines. Furthermore, shRNA-mediated knockdown of HDAC1 or HDAC2 resulted in growth inhibition in these cells. We then assessed a compound that specifically inhibits only HDAC1 and HDAC2. This compound suppressed growth and induced apoptosis in B-ALL cell lines in vitro and in vivo, whereas it was far less effective against other B-cell–derived malignancies. Conclusions: Here, we show that HDAC inhibitors are a potential therapeutic option for B-ALL, and that a more specific inhibitor of HDAC1 and HDAC2 could be therapeutically useful for patients with B-ALL. Clin Cancer Res; 21(10); 2348–58. ©2015 AACR.

FLT3 as a Therapeutic Target in Childhood Acute Leukemia

During the past few years, a major focus of leukemia research has centered on tyrosine kinases as potential therapeutic targets. This is due in large part to the success of imatinib mesylate (STI571, Gleevec), which has proven effective as a therapy for chronic myeloid leukemias bearing the t(9;22) encoding the BCR-ABL kinase. It has become increasingly evident that mutations producing constitutively active tyrosine kinases play a role in leukemogenesis. Another kinase that has drawn significant attention is the FMS-like tyrosine kinase 3 (FLT3). FLT3 is expressed in most childhood acute leukemias. Select genetic subgroups possess particularly high-level expression, with a significant percentage therein harboring activating mutations. In this review we will discuss FLT3 as a potential therapeutic target in childhood acute leukemias. We will highlight the role of FLT3 in hematopoiesis, and how when activated, it may play a role in the development of acute myeloid or acute lymphoblastic leukemia. We will examine the successes in elucidating FLT3 function in acute leukemias, highlight current FLT3 targeted therapeutics, and discuss how FLT3 inhibitors might be used in combination therapies in the future.

Abstract 3523: Discovery of a novel BET inhibitor INCB054329

Bromodomains (BD) are protein modules that bind acetylated lysine residues and are components of many epigenetic modifiers and transcription factors. The BET (Bromodomain and extra-terminal) family is composed of four members each harboring two tandem BDs. BET proteins are critical regulators of transcription through interactions with complexes including Mediator and p-TEFb at gene promoter and enhancer elements. Studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins is therapeutic in models of cancer and acute inflammation. We describe the preclinical activity of a novel BET inhibitor INCB054329 for the potential treatment of malignant diseases. INCB054329 inhibited binding of BRD2, BRD3 and BRD4 to an acetylated histone H4 peptide with low nanomolar potency. In myeloma cell lines, treatment with INCB054329 inhibited expression of c-MYC and induced HEXIM1. The majority of myeloma, AML, and lymphoma cell lines tested were growth inhibited by INCB054329 with potencies less than 200 nM. Selectivity was seen when compared with nontransformed cells as the potency for growth inhibition of IL-2 stimulated T-cells from normal donors was greater than 1300 nM. Cell cycle analysis revealed treatment-induced G1 arrest. Furthermore in both AML and lymphoma cell lines, INCB054329 induced apoptosis consistent with increased expression of pro-apoptotic regulators. In vivo, oral administration of INCB054329 inhibited tumor growth in several models of hematologic cancers. In the MM1.S multiple myeloma xenograft model, inhibition of tumor growth was correlated with reduction of c-MYC levels. PK-PD analysis showed c-MYC suppression was associated with an IC50 value of less than 100 nM in vivo. In summary these studies demonstrate that INCB054329 is a potent inhibitor of BET transcriptional regulators in models of hematologic malignancies in vitro and in vivo and support its clinical development for the treatment of cancer. Citation Format: Phillip CC Liu, Xuesong Mike Liu, Matthew C. Stubbs, Thomas Maduskuie, Richard Sparks, Nina Zolotarjova, Jun Li, Xiaoming Wen, Margaret Favata, Patricia Feldman, Alla Volgina, Darlise DiMatteo, Robert Collins, Nikoo Falahatpisheh, Padmaja Polam, Yu Li, Maryanne Covington, Sharon Diamond-Fosbenner, Richard Wynn, Timothy Burn, Kris Vaddi, Swamy Yeleswaram, Andrew P. Combs, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Discovery of a novel BET inhibitor INCB054329. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3523. doi:10.1158/1538-7445.AM2015-3523

Abstract 692: The BET inhibitor INCB054329 is synergistic with JAK1 inhibition in models of multiple myeloma

Bromodomain and Extra Terminal (BET) protein inhibitors have emerged as a potentially effective therapeutic option for multiple tumor types, through their ability to regulate expression of genes necessary for proliferation and survival. For example, multiple myeloma (MM) cells have been shown to be highly sensitive to BET inhibition due in large part to the ability of BET proteins to control transcription of c-myc, an oncogene known to be dysregulated in MM. Likewise, some inflammatory response and cytokine signaling pathways associated with MM (eg. IL-6/JAK/STAT pathway) have also been shown to be reliant on BET proteins. Therefore, inhibition of both BET proteins and the JAK/STAT signaling pathway may be beneficial to MM patients. Here we assess the in vitro and in vivo effects of combining clinical compounds that target BET proteins and JAK in multiple myeloma cell lines. Studies were performed using the potent pan-BET inhibitor INCB054329 and selective JAK1 inhibitors. When tested in cell proliferation assays, the combination of BET and JAK1 inhibitors displayed strong synergistic effects in the IL-6 dependent INA-6 MM cell line in vitro. Western blots also revealed that several pharmacodynamic (PD) markers including c-MYC, PIM-2 and phospho-STAT3 were further repressed with the combination than with single agents alone. Likewise, the c-MYC and p-STAT3 PD markers could also be increasingly repressed in vivo by combined administration of BET and JAK1 inhibitors in the INA-6 mouse xenograft model. In vivo efficacy experiments in the INA-6 model resulted in enhanced, synergistic tumor growth inhibition in the BET/JAK inhibitor cohort as compared with the single drug cohorts. Interestingly, the cytokine independent MM1.S cell line was also sensitive to the BET/JAK inhibitor combination in vivo, while being far less sensitive to JAK1 inhibition as a monotherapy. In the MM1.S model, the c-MYC and p-STAT3 PD markers also behaved as seen in the INA-6 model. Our data indicate that the pharmacological inhibition of BET proteins and JAK1 yields strong combinatorial effects in MM cell lines both in vitro and in vivo. Therefore, dual inhibition of BET proteins and the JAK/STAT signaling pathway may offer a novel therapeutic approach and suggest a potential clinical utility for this drug combination in MM. Citation Format: Matthew C. Stubbs, Xuesong M. Liu, Xiaoming Wen, Jun Li, Valerie Dostalik, Sybil O9Connor, Eian Caulder, Margaret Favata, Mark Rupar, Yu Li, Beth Rumberger, Thomas Maduskuie, Richard Sparks, Nikoo Falahatpisheh, Padmaja Polam, Kris Vaddi, Timothy Burn, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. The BET inhibitor INCB054329 is synergistic with JAK1 inhibition in models of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 692. doi:10.1158/1538-7445.AM2015-692

The BET inhibitor INCB054329 reduces homologous recombination efficiency and augments PARP inhibitor activity in ovarian cancer

OBJECTIVE Homologous recombination (HR)-proficient ovarian tumors have poorer clinical outcomes and show resistance to poly ADP ribose polymerase inhibitors (PARPi). A subset of HR-proficient ovarian tumors show amplification in bromodomain and extra-terminal (BET) genes such as BRD4. We aimed to test the hypothesis that BRD4 inhibition sensitizes ovarian cancer cells to PARPi by reducing HR efficiency and increasing DNA damage. METHODS HR-proficient ovarian cancer cell lines (OVCAR-3, OVCAR-4, SKOV-3, UWB1.289+BRCA1) were treated with BRD4-targeting siRNA, novel (INB054329, INCB057643) and established (JQ1) BET inhibitors (BETi) and PARPi (olaparib, rucaparib). Cell growth and viability were assessed by sulforhodamine B assays in vitro, and in SKOV-3 and ovarian cancer patient-derived xenografts in vivo. DNA damage and repair (pH2AX, RAD51 and BRCA1 foci formation, and DRGFP HR reporter activity), apoptosis markers (cleaved PARP, cleaved caspase-3, Bax) and proliferation markers (PCNA, Ki67) were assessed by immunofluorescence and western blot. RESULTS In cultured cells, inhibition of BRD4 by siRNA or INCB054329 reduced expression and function of BRCA1 and RAD51, reduced HR reporter activity, and sensitized the cells to olaparib-induced growth inhibition, DNA damage induction and apoptosis. Synergy was observed between all BETi tested and PARPi. INCB054329 and olaparib also co-operatively inhibited xenograft tumor growth, accompanied by reduced BRCA1 expression and proliferation, and increased apoptosis and DNA damage. CONCLUSIONS These results provide strong rationale for using BETi to extend therapeutic efficacy of PARPi to HR-proficient ovarian tumors and could benefit a substantial number of women diagnosed with this devastating disease.