Vulnerabilities In Acute Myeloid Leukemia Research Articles

Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective white blood cell differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity, that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics of a conditional knockout mouse, we demonstrate that Kat2a contributes to leukemia propagation through preservation of leukemia stem-like cells. Kat2a loss impacts transcription factor binding and reduces transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels. Destabilization of target programs shifts leukemia cell fate out of self-renewal into differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and distinct stages of cancer evolution.

Integrating Enhancer Profiling and CRISPR Dropout Screen Revealed Selenoprotein Synthesis Pathway As a New Vulnerability in AML

Alterations of the epigenetic landscape and transcription are hallmarks of acute myeloid leukemia (AML) that drive leukemogenic gene expression and therefore can be exploited for therapeutic intervention. To look for such targets that harbor both an altered epigenetic feature and are genetically essential for AML cells, we performed a multi-database analysis integrating pan-cancer super enhancer landscapes with whole genome CRISPR dropout screens. Among the top targets, we discovered SEPHS2. An enhancer was present upstream of the gene marked by H3K27ac and bound by leukemogenic transcription factors including MYB, Pu.1 and RUNX1. In addition, AML cells with SEPHS2 deletion significantly dropped out in a genome wide CRISPR screen. This gene encodes a critical enzyme in the underappreciated selenoprotein synthesis pathway which was highly upregulated in TCGA AML patients compared to control blood cells from healthy individuals. Collectively, our initial bioinformatic analysis suggested that the selenoprotein synthesis pathway is a new vulnerability in AML. To test the functional requirement of the selenoprotein synthesis pathway in AML and other cells, we performed CRISPR mediated deletion of three key genes in the selenoprotein synthesis pathway, SEPHS2, SEPSECS and EEFSEC. The human AML cell lines (MOLM13, THP1 and Kasumi1), murine AML cells transformed by MLL-AF9 and human AML PDX cells all depicted a significant dependency on these genes, while proliferation of normal cord blood cells and myeloma cells (U266B1) was almost not affected. We then transplanted these cells into recipients. Deletion of SEPHS2, SEPSECS or EEFSEC significantly ameliorated AML progression, indicated by decreased AML burden and extended survival. Since selenoproteins including GPX1 and GPX4 are known antioxidants, we hypothesized that perturbing the selenoprotein synthesis pathway disrupted the redox state in AML cells. We found that deletion of SEPHS2, SEPSECS or EEFSEC elevated ROS in AML cells as demonstrated by Cell-Rox or DCF-DA staining. Western blotting revealed significantly downregulated GPX4 level and upregulated DNA damage marker γ-H2AX. Moreover, the defective proliferation was partially rescued by adding antioxidant TEMPOL. These results suggested selenoprotein synthesis pathway produced key antioxidants to balance the proper redox state and was required for AML cell proliferation. A major source of selenium is diet. Therefore, we hypothesized that consuming selenium low diet could suppress AML. We compared the survival of AML bearing mice on selenium proficient and deficient diet. The selenium deficient diet significantly extended survival, lowered GPX4 level and increased ROS in AML cells. Interestingly, normal mouse on selenium deficient diet for a 3-months period did not develop any abnormalities in CBC or bone marrow hematopoiesis. This suggests selenium deficient diet could be clinically applicable without significant side effects. Altogether, the integration of a pan-cancer enhancer landscape study with CRISPR dropout gene screen offered a powerful tool to dissect cancer targets that possessed unique enhancer features and genetic essentiality. The analysis yielded SEPHS2 and its selenoprotein synthesis pathway to be a new vulnerability in AML. The underappreciated selenoprotein synthesis pathway was key to produce antioxidant selenoproteins such as GPX1 and GPX4 to maintain a proper redox state in AML. Deleting the genes or removing selenium from diet could perturb the pathway and ameliorate the AML disease. Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

Cell Type-Specific Effects of Crizotinib in Human Acute Myeloid Leukemia with TP53 Alterations

Introduction: In acute myeloid leukemia (AML) standard therapies induce complete remission in 50-70% of patients, but overall two-year survival is less than 20-30% because of high relapse rates. AML with TP53 alteration is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplantation. Mutations in TP53 are seen in approximately 20% of AML and confer a particularly poor prognosis decreasing the 1-2 year survival rates to 0-10%. Leukemia stem cells (LSCs) are defined as the cells that recapitulate the leukemia and cause relapse. Recent large-scale drug-screening efforts to map treatment vulnerabilities in AML have revealed that crizotinib can reduce the in vitro viability of unselected primary human AML bone marrow samples with TP53 alterations (Tyner et al. Nature 2018). Crizotinib is a tyrosine kinase inhibitor that targets ALK, MET, and RON and is FDA approved for treatment of lung cancer. Since LSCs are largely responsible for AML mortality, we investigated whether crizotinib targets LSCs in primary, diagnostic human AML samples with TP53 alterations. We used mass cytometry (CyTOF2) to determine the cell-type specific effects of this drug on intracellular signaling states. We profiled treated samples with a panel that includes immunophenotypic markers (including LSC markers) and intracellular signaling molecules implicated in AML pathogenesis and aggressiveness. Results: Primary human AML samples with TP53 alterations were treated with crizotinib (10 mM, n=3). There was a reduction of viability by 48 hours of in vitro treatment with crizotinib, relative to vehicle (mean 64% reduction in viability). Crizotinib abolished all colony formation in semi-solid media. Mass cytometry analysis revealed that crizotinib treatment led to a loss of cells bearing LSC-specific markers such as CD123 and TIM3. Crizotinib treatment also induced a consistent loss of phosphorylated-signaling intermediates of the STAT and p38/MAPAPKII pathways in all samples tested. Notably, crizotinib treatment also induced significant reductions in total and phosphorylated NFKB in all samples tested. Conclusions: These data reveal cell-type specific effects of crizotinib in human AML with TP53 alterations. Crizotinib induced a specific loss of LSCs and inhibition of the STAT, p38/MAPAPKII and NFKB pathways. These data suggest that crizotinib inhibition may target LSCs in AML with TP53 alterations. Figure Disclosures No relevant conflicts of interest to declare.

Efficient Elimination of Acute Myeloid Leukemia Cells through Inhibition of KDM4A in Combination with PARP Inhibition

Epigenetic therapies are emerging as a promising therapeutic strategy for acute myeloid leukemia (AML), exemplified by advances in the development of inhibitors targeting DNMT3A, DOT1L and LSD1. We identified an essential role for the H3K9me3 histone demethylase, KDM4A, in maintaining AML cell survival with genetic depletion of KDM4A having no effect on normal hematopoiesis. Therefore, we hypothesise KDM4A inhibition may represent a novel and effective strategy to treat AML. To address this, we developed a series of novel KDM4A inhibitors (KDM4Ai), based on the structure of pan inhibitor IOX1, and fully characterised their functional potential in AML cells representing major molecular subtypes, and primary patient blasts in comparison with healthy donor cells, as single agents or in combination with other anti-cancer drugs. To evaluate these compounds in physiological conditions, we utilised a stromal co-culture system mimicking the bone marrow microenvironment. Furthermore, we carried out global transcriptomic profiling by RNA-seq to elucidate the molecular consequences responsible for KDM4Ai induced leukemic killing. As a mono-therapy, KDM4Ai induced leukemic cell differentiation and apoptosis in a broad spectrum of human AML cells, with an IC50 of 3.2µM ± 0.2 in MLL-AF9 driven THP1 cells after 48hr treatment (n=3), similar efficacy was observed in other human AML cell lines (n= ≤3) including Kasumi1 (2.69µM ± 0.1) , OCI-AML3 (4.9 µM ± 1.0) and MOLM13 (1.7 µM ± 0.7) and in primary patient blasts (3.8µM). A complete removal of colony forming potential was observed upon treatment (n = 2). The global expression of KDM4A's established substrate, H3K9me3 was upregulated by immunofluorescence and transcriptional changes in a 9-gene signature identified previously as direct KDM4A downstream targets, is indicative of an on-target effect by KDM4Ai. Importantly, KDM4Ai specifically reduced CD34+leukemic stem cell enriched population ( reduced by 6%). In contrast, a non significant reduction was observed on donor CD34+hematopoietic stem and progenitor cells proliferation and apoptosis suggesting a therapeutic window. Cytoprotection provided by stromal co-culture in both AML cell lines and primary samples resulted in a 50% decrease in apoptotic cells with maintenance of the CD34+compartment . Taking these results into account we identify importance of the microenvironment in drug mechanism and resistance. To better understand the mechanism driving this selective anti-leukemic effect, we performed transcriptomic analysis on KDM4Ai treated THP1 cells (n=3). Corroborating the differentiation phenotype, pathway analysis showed an enrichment of IL4 & IL13 signalling (Enrichment score (ES) = - 0.53, q value = 0.025), and neutrophil degranulation (Enrichment score (ES) = - 0.51, q value = 0.025), this was accompanied by significant up-regulation of DNA damage response pathways (ES = 0.66, q value = 0.025) . These results were confirmed in AML cell lines, displaying accumulation > 20% of γH2AX by intracellular flow cytometry and PARP cleavage by western blot following treatment. Based on these results we hypothesised that KDM4Ai may sensitise leukemic cells to DNA damage pathway inhibitors, such as PARP inhibitors (PARPi) (n = ≤3). While standard chemotherapies, such as cytarabine and azacitidine, in combination with KDM4Ai showed a largely additive effect, a dual inhibition of KDM4Ai at 3mM with 5mM olaparib (PARPi), exhibited a Combination Index of ~0.69 with a decrease in proliferation (>15% reduction) and increased apoptosis (>20%) in MLLr-AML cell lines compared with KDM4Ai alone (p=0.005). This effect was corroborated ex vivo using cells isolated from a patient derived xenograft model of MLL-AF10 (n=3). Taken together these results suggest a synergistic leukemic cell killing and have subsequently been subjected to global RNA-seq to confirm the detailed molecular mechanism underlying the synergistic effect. Pharmacological inhibition of KDM4A using novel compounds effectively eliminated leukemic cells sparing normal hematopoietic cells with a synthetic lethality observed through combination with PARPi offering a promising therapeutic strategy in AML. Our data further support the essential role for KDM4A in myeloid oncogenesis, promoting future clinical evaluation of KDM4Ai its associated downstream targets as potential tractable therapeutic vulnerabilities in AML. Disclosures No relevant conflicts of interest to declare.

The GPR68/BCL2 Axis Remodels Metabolism in AML By Relocating Calcium

Hematopoietic stem cells (HSC) mainly engage glycolysis while leukemia stem cells (LSC), such as in acute myeloid leukemia (AML), heavily rely on mitochondrial (Mt) respiration (i.e. oxidative phosphorylation, OxPhos) to fuel energy. Growing evidence suggests this metabolic reprogramming confers therapeutic vulnerabilities in AML. BCL2 is overexpressed in LSC from AML patients, while BCL2 inhibitors, such as venetoclax (VEN), have been shown to suppress OxPhos in LSC and to eradicate LSC. In clinical practice, half of the patients fail to respond to VEN. VEN is a costly medication and patients who are resistant to VEN forgo alternative treatment at that time. Prediction of the response to VEN and strategies to circumvent resistance are urgently needed. BCL2 is shown to increase Mt Ca2+ levels ([Ca2+]m), which enhance OxPhos through activation of the Ca2+-sensitive dehydrogenases within the tricarboxylic acid (TCA) cycle. G protein-coupled receptor 68 (GPR68) is a proton sensor, activating phospholipase C that leads to releasing of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and elevation of cytosolic Ca2+ levels ([Ca2+]c). This prompted us to examine the cooperative effect of GPR68 and BCL2 on the Ca2+/OxPhos pathway in AML, and particularly in LSC. Expression of leukemic oncogenes (i.e. MLL-AF9 and HRASG12D) in mouse hematopoietic stem and progenitor cells, such as Lineage-Sca-1+cKit+ (LSK) cells or granulocyte-monocyte progenitor cells, promote leukemogenesis as evidenced by serial colony formation in vitro and leukemia development in vivo. We found significantly reduced colonies in oncogene-expressing LSK cells from Gpr68 knockout mice compared to wild type mice. Deletion of Gpr68 reduced [Ca2+]c in oncogene-expressing LSK cells. We next examined the function of GPR68 in human AML cell lines. Knockdown of GPR68 with shRNA reduced cell growth and colony formation, and induced apoptosis in AML cells. Knockdown of GPR68 also reduced [Ca2+]c and Mt membrane potential (Δψm) in AML cells, indicating reduced Mt OxPhos. These results suggest that GPR68 regulates the Ca2+/OxPhos pathway in AML cells. We next examined the cooperative effect of GPR68 and BCL2 by jointly inhibiting their activities with pharmacological agents (i.e. GPR68 antibody and VEN, respectively) in AML cell lines and AML patient-derived xenograft models. Of note, the expression of GPR68 was positively correlated with the sensitivity to VEN in AML cells. For AML cells that were resistant to VEN, GPR68 antibody but not an unrelated antibody increased the sensitivity to VEN by enhancing apoptosis, indicating that GPR68 and BCL2 co-regulate AML cell survival. We next examined the mechanism of this synthetic lethality by measuring cellular respiration. Single treatment with VEN reduced Δψm, ATP production and O2 consumption in AML cells. Cotreatment with VEN and GPR68 antibody further reduced Δψm, ATP production and O2 consumption in AML cells, indicating that GPR68 and BCL2 co-regulate Mt OxPhos. Given that GPR68 releases Ca2+ from ER to cytosol, while BCL2 maintains [Ca2+]m by inhibiting its extrusion from Mt, we hypothesize that the GPR68/BCL2 axis relocates Ca2+ from ER to Mt. As expected, treatment with GPR68 antibody reduced [Ca2+]c. Cotreatment with VEN and GPR68 antibody increased [Ca2+]c, indicating enhanced extrusion of Ca2+ from Mt to cytosol by VEN. Consistently, cotreatment with VEN and GPR68 antibody reduced the activity of isocitrate dehydrogenase, the rate limiting enzyme in the TCA cycle, in AML cells. These results indicate that GPR68 and BCL2 co-regulate the Ca2+/OxPhos pathway in AML cells and that co-inhibition of GPR68 and BCL2 may enhance lethality and overcome VEN resistance. In summary, our study suggests that the GPR68/BCL2 axis co-regulates AML cell survival by relocating Ca2+ from ER to Mt thus enhancing Mt OxPhos, and that disruption of the GPR68/BCL2 axis provides a novel therapeutic strategy to overcome resistance to VEN. Disclosures No relevant conflicts of interest to declare.

S-Adenosyl Methionine Synthesis Impacts Maintenance of Acute Myeloid Leukemia Cells with Regulation of Transcriptional Elongation

Epigenetic dysregulation is at the heart of Acute Myeloid Leukemia (AML), and its mechanistic understanding holds promise in improving AML dismal prognosis. Modification of chromatin accessibility through post-translational modification of histones alters gene expression activity and is directly dependent on cell metabolism for generation of the modifying groups. Methionine metabolism generates S-adenosyl methionine (SAM), the universal methyl donor in the cell, through the activity of multimeric methionine adenosyl transferase (MAT) enzymes. MAT2A is an obligatory subunit in hematopoietic cells, and we have recently tabled it in a CRISPR drop-out screen [1] as a candidate vulnerability in AML.We have validated the anti-AML effects of MAT2A ablation using shRNA-mediated gene expression knockdown, and could further phenocopy cell line selectivity observed in our CRISPR screen through the use of the MAT2A selective inhibitor FIDAS-5 [2] (IC50 MOLM-13 1.0μM, OCI-AML3 0.7μM, MV4.11 11μM).MAT2A inhibition significantly depleted SAM levels (fold change to DMSO, FC=0.20±0.07; p<0.05, MOLM-13 cells), and resulted in decreased growth and enhanced apoptosis (MOLM-13 FC=1.34±0.21;p<0.05, OCI-AML3 FC=3.0±0.34; p<0.05, MV4.11 FC=1.09±0.19;p=0.52) of cultured AML cells, without an obvious impact on differentiation. Similar results were observed in AML patient samples (n=11), which displayed reduced capacity to initiate colonies in semi-solid medium (FC=0.47±0.21; p<0.0005) and had hindered expansion in medium-term co-cultures on MS-5 stroma, with accumulation of cells with intermediate, but not high, levels of primitive marker CD34. MLL-AF9 transformed primary mouse bone marrow cells were equally affected (FC colony efficiency=0.05±0.03; p<0.005). Significantly, MAT2A inhibition did not affect colony-formation or multi-lineage differentiation of cord blood-derived CD34+ cells in colony-forming progenitor assays (FC=1.01±0.25p=0.97), indicating selective vulnerability of AML cells.We analysed the consequences of MAT2A inhibition on chromatin methyl modifications, and observed a specific reduction in the active elongation mark H3K36me3. The associated transcriptional programme is currently being investigated. Interestingly, contrary to reports in colon cancer cells upon methionine restriction and reduced SAM levels [3], we did not observe loss of H3K4me3 at active promoters, suggesting a selective AML dependence on transcription elongation.In conclusion, we identify SAM synthesis as a novel vulnerability in AML through regulation of transcriptional elongation. Our findings highlight the therapeutic potential of modulating metabolite availability for epigenetic and transcriptional re-programming in leukemia.

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of Mitochondrial Vulnerability in Acute Myeloid Leukemia

BACKGROUND60% to 70% of Acute Myeloid Leukemia (AML) patients enter complete remission after induction regimen, but the majority relapse within 3 years due to the outgrowth of therapy resistant Leukemia Stem Cells (LSCs). Identification of novel treatment strategies effective against these cells thus represents an outstanding medical need. We developed a cell culture method, which transiently maintains LSC activity ex vivo (Pabst et al., Nature Methods, 2014) and enables chemical interrogation of cell types relevant for the progression of the disease. Overall, HSCs and LSCs share numerous biological traits, making specific LSC eradication challenging. However, striking differences in energy metabolism between normal and leukemic stem cells have recently been suggested. While HSCs appear to rely primarily on anaerobic glycolysis for energy production, LSCs seem to depend on mitochondrial oxidative phosphorylation for their survival. Targeting mitochondrial respiration could therefore represent an effective approach for the specific eradication of LSCs. AIMWe aimed to identify novel therapeutic targets for AMLs with poor treatment outcome. The study relied on the Leucegene approach that integrates results generated by RNA sequencing analysis of primary human AML specimens, detailed clinical and cytogenetic annotations provided by the Quebec leukemia cell bank and ex vivo responses of primary AML samples to various chemical compounds. Our study specifically focused on specimens originating from patients with poor (overall survival < 3 years) and good (overall survival ≥ 3 years) response to standard chemotherapy, and did not include cases of Acute Promyelocytic Leukemia (APL). RESULTSWe identified Mubritinib, previously described as an ERBB2 inhibitor, as a novel anti-leukemic agent, which selectively inhibits the viability of leukemic cells from therapy-resistant AML patients, but does not affect normal CD34+ cord blood cells. Exposure to Mubritinib triggered apoptotic cell death in a subset of AML samples with high mitochondrial function-related gene expression, high relapse rates, and short overall survival. Sensitivity to Mubritinib also strongly associated with the intermediate cytogenetic risk category, normal karyotype (NK), and NPM1, FLT3 (ITD) and DNMT3A mutations. Conversely, resistance to Mubritinib associated with favorable cytogenetic risk AMLs, Core Binging Factor (CBF) leukemias and KIT mutations. Mubritinib has been developed as an ERBB2 kinase inhibitor. Intriguingly, we found that ERBB2 is not expressed in Mubritinib-sensitive AML specimens, suggesting that the anti-leukemic activity of this compound is likely not mediated by ERBB2 inhibition. Using a combination of functional genomics and biochemical analyses, we demonstrated that Mubritinib directly inhibits the mitochondrial Electron Transport Chain (ETC) complex I, which leads to a decrease in oxidative phosphorylation activity and to induction of oxidative stress. The impact of Mubritinib on AML progression was explored using a syngeneic mouse model (MLL-AF9 tdTomato-positive leukemia). Recipients of MLL-AF9 cells treated with Mubritinib exhibited a 19-fold decrease in the number of tdTomato-positive cells in the bone marrow and a 42-fold decrease in the spleens compared to control mice. Short-term treatment also led to a 37% increase in the median overall survival of Mubritinib exposed recipients compared to vehicle treated mice. Importantly, and in agreement with our observation that Mubritinib treatment does not impede proliferation of normal hematopoietic CD34+ cells in vitro, Mubritinib treatment had no impact on the number of non-transduced (tdTomato negative) nucleated bone marrow cells of recipients. CONCLUSIONSWe uncovered the clinical, mutational, and transcriptional landscape of mitochondrial vulnerability in AML and identified Mubritinib as a novel ETC complex I inhibitor with therapeutic potential for approximately 30% of AML cases currently lacking effective treatment options. As Mubritinib completed a phase I clinical trial in the context of ERBB2-positive solid tumors, our work suggests an opportunity to re-purpose Mubritinib's usage for this genetically distinct subgroup of poor outcome AML patients. DisclosuresNo relevant conflicts of interest to declare.

SRPK1 Is a Therapeutic Vulnerability in Acute Myeloid Leukemia through Its Effects on Alternative Isoforms of Epigenetic Regulators Including BRD4

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. Aberrant mRNA splicing plays an important role in cancer development and genes coding for several of the major components of the spliceosome are targeted by somatic mutations in several cancers including myelodysplastic syndromes and AML. Recently, myeloid neoplasms harbouring spliceosome gene mutations were shown to be preferentially susceptible to pharmacological disruption of the spliceosome. Here we report that targeting particular pathways of the spliceosome machinery can also be an effective therapeutic strategy in other types of AML.Recently, we generated a comprehensive catalogue of genetic vulnerabilities in AML using CRISPR-Cas9 genome-wide recessive screens and reported several novel intuitive and non-intuitive therapeutic candidates. Amongst these we identified SRPK1, the gene coding for a serine-threonine kinase that phosphorylates the major spliceosome protein SRSF1. Here, we demonstrate that targeted genetic disruption of SRPK1 in MLL-rearranged AMLs leads to differentiation and apoptosis (Fig. 1A). Additionally, the survival of immunocompromised mice transplanted with human AML cell lines carrying the MLL-AF9 fusion gene, namely MOLM-13 and THP-1, was significantly prolonged by genetic disruption of SRPK1 with CRISPR-Cas9. Similar effects were seen with pharmacological inhibition of SRPK1 in vitro and in vivo, using the novel SRPK1-specific kinase inhibitor SPHINX31 (Fig. 1B-C). Importantly, we go on to demonstrate that, while the SRPK1 kinase activity is required for AML cell survival, it is dispensable for normal hematopoiesis.At the molecular level, we show that genetic or pharmacological inhibition of SRPK1 was associated with widespread changes in the splicing of multiple genes including several with roles in leukemogenesis such as MYB, BRD4 and MED24 . We focused on BRD4 as its splicing isoforms have distinct molecular properties and found that SRPK1 inhibition led to a substantial switch from the short (BRD4S) to the long (BRD4L) isoform at the mRNA and protein levels (Fig. 1D-E). This was associated with BRD4 eviction from genomic loci involved in myeloid leukemogenesis including BCL2 and MYC. Notably, ectopic expression of the short (BRD4S) isoform rescued the phenotype of SRPK1 inhibition suggesting that the observed BRD4 splicing switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Furthermore, we show that the BRD inhibitor iBET-151 synergizes with SRPK1 inhibition to kill human MLL-AF9 -driven AMLs in vitro and in vivo.Collectively our findings reveal that SRPK1 is required for normal splicing of key epigenetic regulators including BRD4 and represents a novel therapeutic vulnerability in AML that can be used alone or in combination with clinically relevant epigenetic drugs to enhance their anti-leukemic effects. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vcp-Regulated Homologous Recombination Represents a New Druggable Vulnerability in Acute Myeloid Leukemia

Mammalian cells have developed sophisticated defense mechanisms to counteract a wide variety of stresses to which they are continuously exposed. These adaptive mechanisms are rewired in cancers, such as acute myeloid leukemia (AML), to permit oncogenic transformation (Luo J et al, Cell, 2009). Using an MLL-AF9 syngeneic mouse model, we performed a pooled in vivo shRNA screen intended to identify novel stress response vulnerabilities in AML. p97 / VCP, an AAA-ATPase protein chaperone known to be involved in protein homeostasis and ER stress, was identified as a top candidate.We first validated AML cell dependency on VCP in vivo in the MLL-AF9 model and in vitro in a panel of human AML cell lines (n=16) and primary patient samples (n=5), using VCP-directed shRNA, overexpression of a VCP dominant negative mutant or a highly selective small-molecule inhibitor of VCP, NMS-873 (Magnaghi P et al., Nat Chem Biol, 2013). The on target effect of NMS-873 in an AML context was validated using a VCP mutant (A530T), which confers resistance to VCP inhibition.We next sought to dissect the molecular mechanism by which VCP is essential to AML cell survival and proliferation. Unexpectedly, we determined that targeting VCP did not impair AML cell viability through alteration of the “proteotoxic stress” response (no accumulation of polyubiquitinilated proteins, no consistent change in proteasomal enzymatic activities and no correlation of NMS-873 sensitivity to bortezomib sensitivity in a panel of 16 AML cell lines). Using a VCP dominant negative mutant unable to translocate into the nucleus, we demonstrated that the inhibition of the nuclear, but not the cytoplasmic, fraction of VCP was sufficient to abrogate leukemic cell viability.To define new potential interacting partners of VCP that could explain its pro-leukemogenic function, we used an immunoprecipitation-mass spectrometry approach in the MV4-11 AML cell line and established by pathway overlapping analysis a significant enrichment of DNA repair pathways among the VCP protein interactome network in AML cells. Further analysis confirmed DNA damage induction through gH2AX accumulation in response to VCP inhibition and a marked reduction of homologous recombination (HR) measured using flow cytometry-based reporter assays. In further support of VCP's role in HR signaling, VCP inhibition blocked activation of the serine/threonine kinase ATM and its direct downstream targets (BRCA1, SMC1, and KAP1) in response to DNA damage induction by etoposide in AML. Indeed, the pattern of sensitivity of a panel of 16 AML cell lines and 16 primary patient samples to an ATM inhibitor, KU-55933, was highly correlated with sensitivity to the VCP inhibitor (Spearman score 0.78 and 0.72, respectively).In conclusion, we identified and validated VCP as a druggable dependency in AML and dissected the mechanistic underpinnings of VCP's role in HR orchestration through activation of ATM. DisclosuresDeAngelo:Amgen: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Shire: Honoraria; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Celgene: Research Funding; BMS: Consultancy; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Incyte: Consultancy, Honoraria; Glycomimetics: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Immunogen: Honoraria, Research Funding. Stone:Janssen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ono: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Seattle genetics: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Sumitomo: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Arog: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Hermine:Hybrigenics: Research Funding; Novartis: Research Funding; Celgene: Research Funding; INatherys: Equity Ownership, Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Stegmaier:Novartis: Consultancy, Research Funding.

Mutant nucleophosmin1 (NPM1) Links Protein Translation with Oncogenic Metabolism in Leukemia

Nucleophosmin 1 (NPM1) is the most common gene mutated in acute myeloid leukemia (AML). Several putative mechanisms for the role of NPM1 in the generation of AML have been proposed, including acting as a tumor suppressor by promoting the degradation of p53 and sequestration of wild-type NPM1. However, the specificity of the mutation- most commonly a 4 base pair duplication in the C-terminus- suggests that it may be a gain- of- function mutation conferring an unidentified pro-leukemic effect. Using a novel set of leukemia cell lines generated with CRISPR/Cas9 technology in combination with studies in primary hematopoietic cells, we show that mutant NPM1 may promote leukemia by deregulating protein translation with downstream effects on cellular metabolism. Thus, mutant NPM1 may serve as a critical lynchpin to match metabolic precursors and cellular energetics to ribosomal output. The implications of our results suggest that combined metabolic and protein translation inhibition may be a previously unidentified vulnerability in AML with mutated NPM1.CRISPR/Cas9 technology was used to generate a set of isogenic cell lines derived from the heterozygous OCI-AML3 line. Transcriptomic analysis of clones with either parental allele configuration (wild-type and mutant, WTAM) vs. inactivation of the mutant allele (wild-type only, WTO) confirmed that mutant NPM1 regulates HOX genes, as shown in both mouse models and patient samples. Next, the effects of mutant NPM1 on ribosome biogenesis and protein translation were assayed with pulse- chase and static experiments and showed both ribosomal RNA and protein translation were increased in WTAM clones vs. WTO clones. During these investigations, cell size was noted to be increased in WTAM cells. Cell size can be a consequence of the metabolic activity of a cell. Analysis of the rate of glucose consumption and lactate production showed that WTAM cells have increased aerobic glycolysis compared with WTO cells. To test if mutant NPM1 is sufficient to drive both protein translation and aerobic glycolysis in a normal cell, these studies were replicated in normal human hematopoietic stem cells stably expressing either wild-type NPM1 or mutant NPM1.In summary, our results suggest that mutant NPM1 acts through dysregulation of protein translation and participates in metabolic reprogramming required to drive oncogenic transformation. DisclosuresNo relevant conflicts of interest to declare.

Phase I/IB Study of Azacitidine and Hedgehog Pathway Inhibition in Myeloid Malignancies

Background: Hypomethylating agents (HMA) such as 5-Azacitidine (Aza) have overall responses rate of ~35-50% in acute myeloid Leukemia (AML) and higher risk myelodysplastic syndrome (MDS) patients (pts). However, eventually all pts develop resistance on therapy. To improve upfront response rates and to address HMA failure, we performed genome-scale RNAi screens targeting genes on chromosomes 5 and 7, recurrent cytogenetic abnormalities in AML/MDS. These screens identified Hedgehog pathway (Hh) genes as potentially targetable vulnerabilities with Aza. Combination experiments with smoothened (SMO) inhibitors (SMO is a central activating gene within the Hh pathway) showed pre-clinical synergy. These and additional published data support the Hh pathway as a targetable vulnerability in AML, and provide a rational for a trial.Methods: A phase I/Ib protocol of the oral SMO inhibitor LDE225 (Sonidegib) in combination with Aza in untreated and relapsed/refractory (rel/ref) AML, chronic myelomonocytic leukemia (CMML), MDS, or MDS/myeloproliferative neoplasm overlap pts. Objectives of the phase I part are to determine safety, toxicity and MTD and for the phase Ib part to assess the preliminary overall efficacy. Sonidegib was started at dose level (DL) 0 at 400mg oral daily continuously and Aza at the approved doses (75mg/m2 x 7 days or days 5-2-2) with dose adjustment per label. A classical 3+3 design was used and DLT (CTCAE v4.0) defined as > grade 3 CPK elevation or hepatic or non-hematologic adverse events (AEs) lasting > 7 days or any grade 4 event, except for hematologic toxicities. DLT was assessed after 42 days when steady-state concentrations of Sonidegib were reached. Pharmacokinetic assessments were not performed for this study.Results:Patients and Dose Levels: Between 5/22/2014 and the data cut-off date (7/20/2015), 36 pts were enrolled. Median age was 72 y (range 52-85 y) and 64% were male. Most AML pts (23 of 24) had intermediate or unfavorable cytogenetics and most MDS/CMML pts were in the int.- to high-/very high-risk group (IPSS-R). Six pts were treated at DL-0 of Sonidegib 400mg/day and one DLT of CPK elevation was observed. Another patient had grade 4 neutropenia (which at the time was included in the DLT definition, later removed). Due to overall tolerance of the combination at DL 0 and investigator assessment, despite only 1/6 pts having a formal DLT, DL -1 at Sonidegib 200mg was explored at which 0/3 pts (1 pt non-evaluable) had DLT and the phase Ib portion was opened at DL -1. To date a total of 24 AML (13 newly dx, 11 rel/ref), 11 MDS/CMML (5 newly dx, 6 rel/ref ) and 1 myelofibrosis pt have been enrolled.Safety and Tolerability: An initial safety and efficacy assessment for 25 of the 29 pts treated on the phase Ib (n=26) + the MTD cohort of 200 mg (n=3) and for who at least 2 cycles of treatment follow up was available, showed that all pts experienced at least one grade-3 or 4 hematologic AE with a lower rate in the untreated vs. treated cohort. Other, non-hematologic grade-3 and 4 AEs respectively were seen in 24 and 28% of patients-most commonly hyponatremia and fatigue, known AEs for this drug class. Treatment delays occurred in 28% of pts and Aza or Sonidegib dose reductions in 16%. 23 (79%) pts are alive and 6 have died (21%), with one death while pt was actively treated but was deemed unrelated; 11 pts remain on study, with the remainder showing disease progression (n=3) or taken-off study for other reasons (n=3 each for AEs and did not want further treatment; n=4 other).Efficacy: As of 7/20/15 with a median treatment duration of 2 cycles, 2/5 untreated MDS/CMML pts achieved CR/CRi; 1 HMA rel/ref AML had CR; 1 untreated AML pt each had PR and MLFS. 90% of pts have not progressed. Updated results will be presented at the ASH 2015 meeting; by then all 29 pts will have completed a minimum of 4-6 cycles on study and full efficacy and AE result on all pts will be reported. To date the OS at 6 mo is 77% (95% CI: 44-97%) with a median PFS of 4 mo (range: 2-NR). Many pts in the untreated cohorts have just enrolled.Conclusion: Hh pathway inhibition with the clinically well tolerated SMO inhibitor Sonidegib in combination with Aza in MDS and AML pts is well tolerated with mostly hematologic AEs as expected with early signs of clinical activity in untreated and HMA-failure pts. Full efficacy results of 29 pts and updated safety data will be reported for this first combination trial of a SMO inhibitor in combination with a HMA in MDS and AML pts. DisclosuresMesa:Gilead: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; NS Pharma: Research Funding; CTI Biopharma: Research Funding. Al-Kali:Novartis: Research Funding.

Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells.

To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress

AbstractMitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.