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MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/β-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of β-Catenin in a p53-independent manner. Wnt/β-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/β-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/β-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Deregulation of the BCL-2 family interaction network ensures cancer resistance to apoptosis and is a major challenge to current treatments. Cancer cells commonly evade apoptosis through upregulation of the BCL-2 anti-apoptotic proteins; however, more resistant cancers also downregulate or inactivate pro-apoptotic proteins to suppress apoptosis. Here, we find that apoptosis resistance in a diverse panel of solid and hematological malignancies is mediated by both overexpression of BCL-XL and an unprimed apoptotic state, limiting direct and indirect activation mechanisms of pro-apoptotic BAX. Both survival mechanisms can be overcome by the combination of an orally bioavailable BAX activator, BTSA1.2 with Navitoclax. The combination demonstrates synergistic efficacy in apoptosis-resistant cancer cells, xenografts, and patient-derived tumors while sparing healthy tissues. Additionally, functional assays and genomic markers are identified to predict sensitive tumors to the combination treatment. These findings advance the understanding of apoptosis resistance mechanisms and demonstrate a novel therapeutic strategy for cancer treatment.

Doxorubicin remains an essential component of many cancer regimens, but its use is limited by lethal cardiomyopathy, which has been difficult to target, owing to pleiotropic mechanisms leading to apoptotic and necrotic cardiac cell death. Here we show that BAX is rate-limiting in doxorubicin-induced cardiomyopathy and identify a small-molecule BAX inhibitor that blocks both apoptosis and necrosis to prevent this syndrome. By allosterically inhibiting BAX conformational activation, this compound blocks BAX translocation to mitochondria, thereby abrogating both forms of cell death. When co-administered with doxorubicin, this BAX inhibitor prevents cardiomyopathy in zebrafish and mice. Notably, cardioprotection does not compromise the efficacy of doxorubicin in reducing leukemia or breast cancer burden in vivo, primarily due to increased priming of mitochondrial death mechanisms and higher BAX levels in cancer cells. This study identifies BAX as an actionable target for doxorubicin-induced cardiomyopathy and provides a prototype small-molecule therapeutic.

Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell–focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation. High-resolution sequencing of longitudinal patient samples reveals subclonal mutational diversity in the stem cell compartment driving parallel evolution patterns in acute myeloid leukemia progression.

IL1RAP is an emerging target for AML therapy. Studying its cell-intrinsic function revealed that IL1RAP interacts with and amplifies signaling through c-KIT and FLT3 in AML cells. This novel promiscuous role of IL1RAP in AML has implications for therapeutic targeting., The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease., Graphical Abstract

Despite the identification of several oncogenic driver mutations leading to constitutive JAK–STAT activation, the cellular and molecular biology of myeloproliferative neoplasms (MPN) remains incompletely understood. Recent discoveries have identified underlying disease-modifying molecular aberrations contributing to disease initiation and progression. Here, we report that deletion of Nol3 (Nucleolar protein 3) in mice leads to an MPN resembling primary myelofibrosis (PMF). Nol3−/− MPN mice harbor an expanded Thy1+LSK stem cell population exhibiting increased cell cycling and a myelomonocytic differentiation bias. Molecularly, this phenotype is mediated by Nol3−/−-induced JAK–STAT activation and downstream activation of cyclin-dependent kinase 6 (Cdk6) and Myc. Nol3−/− MPN Thy1+LSK cells share significant molecular similarities with primary CD34+ cells from PMF patients. NOL3 levels are decreased in CD34+ cells from PMF patients, and the NOL3 locus is deleted in a subset of patients with myeloid malignancies. Our results reveal a novel genetic PMF-like mouse model and identify a tumor suppressor role for NOL3 in the pathogenesis of myeloid malignancies.

The p53 inhibitor MDMX is overexpressed in the vast majority of patients with AML; however, a causative role of MDMX in leukemogenesis has not yet been investigated. Mdmx transgenic mice (Mdmx-Tg) displayed increased number, proliferation and competitive advantage of hematopoietic stem cells compared to WT littermates. Crossing Mdmx-Tg with preleukemic mouse models representing frequent alterations in human AML, we generated three distinct compound models. We found that Mdmx overexpression triggered progression of each of the different chronic/asymptomatic preleukemic conditions to overt AML, and thereby markedly shortened survival. RNA sequencing revealed that Mdmx overexpression exerted this function through activation of Wnt/b-Catenin signaling in pre-LSC. Mechanistically, we found that MDMX bound CK1a, a canonical degrader of b-Catenin, and led to accumulation and nuclear translocation of b-Catenin. WNT/b-Catenin inhibitors or exogenous overexpression of CK1a were both effective in reversing Mdmx-induced pre-LSC properties, and showed further synergism in combination with MDMX inhibitor treatment. Competitive advantage and accumulation of b-Catenin upon Mdmx overexpression were still seen upon crossing into a p53-/- background, demonstrating that Mdmx-driven WNT/b-Catenin activation and its functional consequences are, at least part, independent of p53. Lastly, upregulation of WNT/b-Catenin pathways correlated with MDMX overexpression in large cohorts of patients with MDS and was associated with increased risk of progression to AML and poor survival. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML switch mechanism effective in different genetically-driven disease subtypes, and reveals WNT/b-Catenin as a novel, non-canonical p53-independent MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Eltrombopag (EP), a small molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy in stimulating sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. Although the functional and molecular effects of EP on megakaryopoiesis have been studied in the past, mechanistic insights into its effects on the earliest stages of hematopoiesis have been limited. Here, we investigated the effects of EP treatment on hematopoietic stem cell (HSC) function using purified primary HSCs in separation-of-function mouse models, including a TPO receptor-deficient strain, and stem cells isolated from patients undergoing TPO-R agonist treatment. Our mechanistic studies showed a stimulatory effect on stem cell self-renewal independently of TPO-R. Human and mouse HSCs responded to acute EP treatment with metabolic and gene expression alterations consistent with a reduction of intracellular labile iron pools that are essential for stem cell maintenance. Iron preloading prevented the stem cell-stimulatory effects of EP. Moreover, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients undergoing therapy with romiplostim, another TPO-R agonist lacking an iron-chelating ability. Taken altogether, our study demonstrates that EP stimulates hematopoiesis at the stem cell level through iron chelation-mediated molecular reprograming and indicates that labile iron pool-regulated pathways can modulate hematopoietic stem cell function.

Modest transcriptional changes caused by genetic or epigenetic mechanisms are frequent in human cancer. Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces acute myeloid leukemia (AML) in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet, moderate PU.1 inhibition is common in AML patients. We assessed functional consequences of modest reductions in PU.1 expression on leukemia development in mice harboring DNA lesions resembling those acquired during human stem cell aging. Heterozygous deletion of an enhancer of PU.1, which resulted in a 35% reduction of PU.1 expression, was sufficient to induce myeloid-biased preleukemic stem cells and their subsequent transformation to AML in a DNA mismatch repair-deficient background. AML progression was mediated by inhibition of expression of a PU.1-cooperating transcription factor, Irf8. Notably, we found marked molecular similarities between the disease in these mice and human myelodysplastic syndrome and AML. This study demonstrates that minimal reduction of a key lineage-specific transcription factor, which commonly occurs in human disease, is sufficient to initiate cancer development, and it provides mechanistic insight into the formation and progression of preleukemic stem cells in AML.

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these inter-actions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled a-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell-enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

Resistance to apoptosis is a hallmark of human cancer. The BCL-2 protein family includes both pro- and anti-apoptotic proteins and has a central role in regulating mitochondrial apoptosis. Cancer cells most frequently ensure their survival and resistance to treatments by overexpression of anti-apoptotic BCL-2 proteins to maintain BAX and other pro-apoptotic members suppressed. Pro-apoptotic BAX is the cardinal executioner BCL-2-family member that, upon conformational activation and oligomerization at the mitochondrial outer membrane (MOM), causes permeabilization of the MOM and release of mitochondrial factors, e.g., cytochrome c, that activate the caspase cascade of apoptosis. Interestingly, the vast majority of cancer cells contain functional BAX in an inactive conformation or suppressed by anti-apoptotic proteins; mutations or alterations in BAX that may cause its inactivation to occur at a low frequency. Therefore, we hypothesized that induction of apoptosis by direct activation of BAX offers the possibility of a new anticancer strategy. Using a structure-based drug design approach we developed BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site (trigger site) and induces conformational changes to BAX, resulting in BAX mitochondrial translocation, oligomerization and mitochondrial dysfunction (Reyna et al., Cancer Cell 2017). BTSA1 induces activation of cytosolic BAX, leading to prompt and robust mitochondrial apoptosis in acute myeloid leukemia (AML) cell lines. The efficacy of BTSA1 is regulated by the availability of anti-apoptotic BCL-2 proteins to inhibit activated BAX and therefore, higher levels of cytosolic BAX monomer correlated with higher efficacy of BAX-mediated mitochondrial dysfunction. BTSA1-induced BAX activation promotes effectively apoptosis in primary AML samples and has significant antitumor activity in human AML xenograft models, increasing host survival. BTSA1 is orally bioavailable with excellent pharmacokinetics at therapeutically effective doses; it does not show any detectable toxicity in the hematopoietic system or other tissues. Importantly, BTSA1 is also effective in inducing apoptosis in leukemic stem cell-enriched fractions (CD34+CD38−) while sparing healthy counterparts. Consistent with their sensitivity to direct BAX activation, AML blasts and highly purified stem and progenitor cell populations from AML patients when compared to healthy counterparts displayed higher expression of BAX. Overall, our data provide proof of concept for BAX as a druggable target and demonstrate the therapeutic potential of direct BAX activation by BTSA1 as an effective treatment strategy in AML. Citation Format: Denis Reyna, Thomas Garner, Andrea Lopez, Felix Kopp, Gaurav Choudhary, Ashwin Sridharan, Swathi-Rao Narayanagari, Kelly Mitchell, Baoxia Dong, Boris Bartholdy, Loren Walensky, Amit Verma, Ulrich Steidl, Evripidis Gavathiotis. Direct small-molecule BAX activation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 951.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells. We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments. The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition. To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival. To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. Disclosures Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl: Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML. We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice. We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers. Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics. 1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) Disclosures Guerlavais: Aileron: Employment. Annis: Aileron Therapeutics: Employment. Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado: Aileron: Employment. Steidl: Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.

Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G1 phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival". Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers. Disclosures Gao: GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment.

Genomic studies have shown that human cancer is rarely associated with a complete loss of transcripts; instead, acquired DNA alterations often occur within the non-coding part of the genome, are enriched in gene-regulatory regions, and cause only moderate transcriptional changes. It is currently not well understood how such moderate gene expression changes impact normal tissue function and how they contribute to malignant transformation. Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) develop through a multi-step transformation process originating in hematopoietic stem cells (HSCs) and mainly present in the elderly (median age of >65 years at diagnosis). Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces AML in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. At the example of the Ets-family transcription factor PU.1, which is indispensable for HSC function and the differentiation of cells within the myeloid as well as lymphoid lineages, we tested the hypothesis that even moderate gene expression alterations of key regulators can drive malignant transformation. We assessed the effects of minimal PU.1 inhibition on hematopoiesis in a novel mouse model that co-models the genomic context found in aging human individuals and patients with MDS/AML. Mice lacking Msh2, the key component of the MutSα and MutSβ complexes mediating DNA mismatch repair, accumulate elevated numbers of point mutations, in particular C/G>T/A transitions and small insertions/deletions resembling the mutation spectrum acquired in HSCs in aging human individuals and patients with MDS and AML. We crossed Msh2-/- mice with animals carrying a heterozygous deletion of an upstream regulatory element of PU.1 (UREΔ/+). UREΔ/+Msh2-/- mice exhibited a significant, but very modest reduction of PU.1 expression on average by 26-37% in fractionated hematopoietic multipotent stem and myeloid progenitor cells. Strikingly, this minimal reduction of PU.1 led to the emergence of an aggressive, transplantable AML in more than two thirds of UREΔ/+Msh2-/- mice which was never observed in URE+/+Msh2-/- mice. Overt leukemia was preceded by a preleukemic phase hallmarked by an expanded population of multipotent murine hematopoietic stem cell enriched cells (HSPCs) that was myeloid-biased and less quiescent than their wild type counterpart. Longitudinal monitoring of preleukemic UREΔ/+Msh2-/- mice revealed a progressive increase in immature myeloid cells along with a gradual decrease in mature myeloid cells, as well as expansion of phenotypic HSPC compartments and multi-lineage dysplasia resembling human MDS. AML progression was accompanied by additional inhibition of a PU.1-cooperating factor, interferon responsive factor 8 (Irf8). Irf8 expression restoration rescued impaired expression of genes harboring PU.1/IRF consensus binding sites, led to the loss of aberrant self-renewal, promoted myeloid differentiation, and induced apoptosis in leukemic UREhetMsh2-/- cells demonstrating that Irf8 impairment functionally cooperates with minimally reduced PU.1 expression in our model. We also found evidence of disease-relevant joint PU.1/IRF8 inhibition in human myeloid leukemogenesis: (1) patients with MDS with a higher risk for the progression to AML had lower IRF8 levels; (2) lower IRF8 expression was detected specifically in AML patients with reduced PU.1 levels; (3) restoration of IRF8 expression induced differentiation in IRF8 low expressing AML cells, and (4) a positive correlation of PU.1 and IRF8 expression was found in human leukemia stem cells, but not in healthy HSCs. Strikingly, comparative pathway analysis revealed a genome-wide molecular resemblance of preleukemic and leukemic UREΔ/+Msh2-/- mice with gene expression profiles from human MDS and AML patients, respectively. Our study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in MDS and AML. Disclosures No relevant conflicts of interest to declare.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients' cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients' cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.

Cell cycling is a tightly regulated process involving the structured expression modulation of various regulatory genes. This process is crucial for the maintenance of cell survival/proliferation in both normal and malignant hematopoietic cells. We have previously described the highly expressed CIITA-BX648577 gene fusion (Steidl C. et al., Nature 2011), involving the novel gene locus BX648577/FLJ27352 /hypothetical LOC 145788/C15orf65 and the Class II Transactivator (CIITA) in the Hodgkin’s lymphoma cell line KM-H2. While CIITA is well known to be involved in the regulation of immune responses, specifically through regulation of the Major Histocompatibility Complex (MHC)-II, nothing is known about the expression and function of the BX648577 locus. The objective of the current study was to (I) study RNA and protein expression of the putative full length gene encoded by the BX648577 (TIHL) gene locus, and (II) study its biological function in normal and malignant hematopoietic cells, including its effects on cell proliferation, clonogenicity and cell death. We detected robust endogenous TIHL RNA and protein expression in a variety of healthy and malignant hematopoietic cell types using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis using a TIHL-specific antibody. At the functional level, we found that ectopic expression of the highly conserved full length TIHL protein in human NB4 leukemia cells and murine hematopoietic progenitor HPC-7 cells leads to enhanced clonogenicity and increased proliferative capacity with significant increases in the percentage of cells in S-phase of the cell cycle. Furthermore, we observed more aggressive leukemia and decreased survival of NSG mice following retro-orbital transplantation of TIHL-expressing compared to empty control-expressing NB4 cells. Interestingly, although we did not observe a change in the rate of cell proliferation or colony forming ability following TIHL overexpression in the ATRA-resistant cell line NB4.306, there was a significant alteration in its cell cycle distribution, with an increase in the fraction of cells in S-phase. The increase in S-phase cells was confirmed by 5-ethynyl-2’-deoxyuridine (EdU) incorporation assays and flow cytometry. Similarly, knockdown of TIHL using 2 independent lentiviral shRNAs, led to a significant decrease in the growth of both NB4 and acute myeloid leukemia KG1a cells in both suspension cultures and semi-solid media. Although we observed slightly increased apoptosis upon TIHL downregulation, the changes could be more significantly attributed to a decrease in the percentage of cells in S-phase within 2-3 days after transduction with the lentiviral shRNAs. Finally, in silico analysis of the TIHL promoter identified various predicted transcriptional regulators of TIHL, the majority of which are cell cycle specific transcription factors including Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), the ets domain transcription factors ELF5 and ELF1, and Glioma-Associated Oncogene Homolog 1 (GLI-1). Our findings thus far strongly support a novel role for TIHL in cell cycle regulation/modulation in both normal and malignant hematopoiesis. Future directions include gene expression studies to identify downstream targets of TIHL following overexpression and knockdown and co-immunoprecipitation coupled to mass spectrometry analysis will be used to identify direct interacting protein partners of this novel gene. Disclosures: No relevant conflicts of interest to declare.