Abstract: Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor survival. Recurrent genetic aberrations, such as chromosomal rearrangements and gene mutations, are currently used for patient classification and prognosis, and form the basis of our current understanding of pathogenic mechanisms. However, these markers incompletely predict disease behavior and outcomes. Alterations in DNA methylation patterns are a major hallmark of cancer and recent studies have demonstrated differential global DNA methylation patterns among AML patients. Here we sought to define a novel approach to classify AML patients using genome-wide DNA methylation patterns and to uncover the biological basis and features associated with these epigenetic patterns by integrating mutation, gene expression, and functional data. Methods We analyzed genome-wide DNA methylation data from Illumina arrays on 649 AML cases combined from the Beat AML consortium housed at the Oregon Health & Science University (OHSU) (n=226), The Ohio State University (n=27), TCGA (n=190), and other published studies (n=206). Small molecule inhibitor response data was obtained from the Beat AML consortium. RNA sequencing and survival analyses were performed by Kaplan-Meier analysis in the TCGA and OHSU cohorts. Results K-mediods based clustering on the 500 most-variable CpGs identified 13 distinct subtypes, we termed AML 'epitypes' (Fig. 1A). Comparative analyses incorporating normal hematopoietic cells revealed that AML epitypes retain unique combinations of biological features including the degree of myeloid development, chromatin landscapes, transcription factor binding and gene expression patterns/pathways. Eleven of 13 epitypes were associated with somatic genetic aberrations, i.e. mutations (or combinations thereof) in NPM1, IDH1/2, TET2, DNMT3A, CEBPA, and chromosomal rearrangements t(8;21), t(15;17) and inv(16) accounted for the majority of patients within each respective epitype. The remaining two epitypes were not associated with dominant genetic events, rather they clustered closely with normal CD34+ hematopoietic stem and progenitor cells. These 'stem-like' epigenetic patterns accounted for 27% of patients and showed limited correlation to other leukemic stem cell gene expression signatures. Stem-like epitypes demonstrated broad resistance to a range of small molecule inhibitors. Differential gene expression analysis between normal cells and epitypes revealed enrichment of the JAK-STAT pathway along with other inflammatory pathways, such as interleukin and interferon signaling, selectively in the stem-like epitypes. Patients with stem-like epitypes exhibited decreased overall survival relative to others independent of age (P & lt;0.0001; Fig. 1B). We next investigated the impact of STAT pathway activation on AML epigenetic patterns. FLT3 mutations are known to selectively activate the STAT pathway in AML and are associated with inferior outcomes. Controlling for the distribution of FLT3 mutations across epitypes, we uncovered a hypomethylation signature enriched in STAT transcription factor binding and FLT3 mutations which we have termed the 'STAT hypomethylation signature' (SHS). SHS-positivity was not restricted to FLT3 mutations, as 34% of SHS+ AMLs lacked FLT3 mutations and, conversely, SHS was absent in 24% of FLT3 mutated patients. SHS-positivity did not impact outcome in the stem-like epitypes; however, within non-stem-like epitypes, SHS+ patients displayed significantly poorer outcomes (P=0.021; Fig 1C). Conclusion Applying our integrative approach of using global DNA methylation profiles to subclassify AML patients, we have uncovered new insights into potential pathogenic mechanisms by which genetic and epigenetic aberrations may contribute to discrete avenues of AML development. Distinct global DNA methylation patterns are commonly driven by genetic aberrations, while others were associated with stem cell-like features and inflammatory pathways. The identification of JAK/STAT pathway upregulation in the stem-like epitype and a subset of non-stem-like samples highlights that nearly half of AML patients exhibit evidence of JAK/STAT activation and experience poor outcomes. Disclosures Druker: Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Beat AML LLC: Other: Service on joint steering committee; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Gilead Sciences: Other: former member of Scientific Advisory Board; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees . Tyner:Petra: Research Funding; Janssen: Research Funding; Aptose: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Takeda: Research Funding; Syros: Research Funding; Array: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Syros: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Agios: Research Funding; Petra: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Agios: Research Funding. Byrd:Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau.

Abstract: Epigenetic alterations are universal in cancer and are important in establishing the malignant phenotype. Dissection of the factors that shape the tumor-specific epigenome may reveal insight into key aspects of tumorigenesis and therapeutic resistance. In chronic lymphocytic leukemia (CLL), we have previously found that broad changes in epigenetic patterns co-occur with the evolution of genetic alterations. We have also uncovered that aberrant patterning of DNA methylation in CLL involves excessive activity of a defined group of transcription factors (TFs), including the early growth response (EGR) TF family. Recent work has further revealed that recurrent mutations in EGR2 are associated with exceptionally poor clinical outcomes in CLL. The basis for the adverse association of EGR2 mutations in CLL is unclear. To explore the role of EGR2 mutations in CLL, we initially performed genome-wide DNA methylation analysis using Illumina arrays on CLL patients harboring EGR2 mutations (n=27) compared to EGR wild-type cases (n=265). We found that the three most common recurrent mutations, occurring at amino acid positions E356K, H384N and D411H within the DNA binding domain, are each associated with an exclusive subset of tumor-specific hypomethylated CpG sites. A search for TF sequence motifs at these loci revealed a strong enrichment of novel derivative EGR2 motifs that differ only marginally (usually by a single nucleotide) from the canonical EGR2 recognition sequence. Each recurrent mutation led to specific enrichment of a different derivative EGR2 motif. Furthermore, the canonical (wild-type) recognition sequence was not enriched, suggesting that mutations re-localize binding activity to derivate sequence motifs rather than simply altering binding affinity. Luciferase enhancer, proximity ligation and electrophoretic mobility shift assays confirmed that each EGR2 mutant protein specifically binds and enhances transcriptional activity only when the matched EGR2 derivative recognition motif is present. These results establish that derivative motif sequences may function as novel cryptic enhancers in the presence of the cognate EGR2 mutant TF. We performed multiomics profiling (DNA methylome, ATAC-seq, ChIP-seq and RNA-seq) to examine the nature of the epigenetic reconfiguration and the phenotypic impact of individual EGR2 mutations. Whole genome bisulfite sequencing of E356K- and H384N-mutated CLL samples (n=4 each) was used to reveal the full complement of recurrent differentially methylated regions (DMRs) across the genome, and recapitulated the mutually-exclusive pattern of DMRs between mutations. Overlaying DMRs with data from ChIP-seq and ATAC-seq experiments in the same samples revealed the nature of EGR2 mutation-specific chromatin reconfiguration to be remarkably mutation-specific. For E356K, hypomethylated DMRs are often associated with foci of accessible chromatin, EGR2 binding, and flanked by gains of H3K4me1 and H3K27ac, indicative of the acquisition of active enhancer function. Conversely, H384N mutations generated fewer DMRs and mainly directed the deposition of H3K4me1 only, indicative of gain of poised enhancers at these loci. RNA-sequencing analyses revealed that a subset of epigenetically reconfigured regions was associated with mutation-specific altered gene expression, and differences were virtually always associated with proximal gene activation. E356K and H384N mutations displayed highly differential gene expression patterns, with E356K exhibiting a greater impact on gene expression. Integrated analyses indicated that E356K mutations may specifically involve activated Notch signaling, revealed by the aberrant activation of Notch target genes and the mutual exclusivity of NOTCH1 mutations, further highlighted by enriched co-mutation of NOTCH1 in H384N-mutated CLL. Together these findings provide an exceptional example of the precise role that a singular TF may play in programming the epigenetic landscape. As there are no known TFs that naturally bind derivative EGR2 motifs, these mutant proteins provide insight into aberrant enhancer generation and the phenotypic impact of (re)directed TF binding in a human disease setting. Although these recurrent mutations are presently only known in CLL, these findings provide insight into the mechanisms that may surround other gain-of-function TF activity in various malignancies. Disclosures Kipps: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees.

Abstract: Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF–induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5nM) and MM patients. It decreased SDF-1–induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P 〈 .03) and MM cell–induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.

Abstract: Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

Abstract: It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Abstract: PKCα and PKCθ cooperate in T-cell alloresponses, which contribute to GVHD. Pharmacologic inhibition of PKCα and PKCθ prevents GVHD and largely preserves GVL responses.

Abstract: It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Abstract: microRNA-155 (miR-155) is a short non-coding RNA that is associated with aggressive cancers and known to promote leukemogenesis. Recently, we have reported that aberrant miR-155 upregulation independently identifies high-risk cytogenetically normal AML patients, suggesting that this miR may also serve as a novel therapeutic target in AML. We and others have shown that miR-155 is positively regulated by NF-kB, a transcription factor that is constitutively activated in leukemic blasts and contributes to their aberrant proliferation and survival. MLN4924 (Millennium Pharmaceuticals Inc) is a novel drug that blocks neddylation and subsequent degradation of the NFkB inhibitor, IkBa, thereby inhibiting translocation of NF-kB to the nucleus. MLN4924 has demonstrated promising activity in early clinical trials for AML. We postulated that downregulation of miR-155 via NF-kB inhibition is at least in part responsible for the antileukemic activity of MLN4924. Methods AML cell lines and primary blasts were treated with 100-1000nM MLN4924 for 3-72 hrs. Messenger RNA and protein levels were determined by quantitative RT-PCR and immunoblotting, respectively. NF-kB activity was measured by luciferase reporter assays. Binding of NF-kB to the miR-155 promoter was detected by electromobility shift assay and Chromatin Immunoprecipitation. Transfection of miR-155 was performed using the siPORT TM NeoFXTM method. Apoptosis was assessed by Annexin V staining. For in vivo studies, we used NOD/SCID/g mice engrafted with MV4-11 cells. Two weeks after transplantation, the engrafted mice received intraperitoneal treatments of 180 mg/kg of MLN4924 every other day for 21 days. Mice in the control group were treated similarly with the vehicle alone (20% 2-hydroxypropyl-betacyclodextrin). Results In AML cell lines and primary AML patient blasts 12hr treatment with MLN4924 resulted in a ∼50% decrease of miR-155 expression at 300nM in THP-1 and MV4-11 cells and at 500nM in AML blasts (p 〈 0.01). This was concomitant with a ∼ 50% and 70% decrease in NF-kB activity and binding to miR-155 promoter, respectively (p 〈 0.01). These results correlated with a significant upregulation of mRNA levels of the key miR-155 target gene, SHIP1 [6-fold (p 〈 0.05), 9-fold (p 〈 0.01), and 2-fold (p 〈 0.05) in THP-1 cells, MV4-11 cells, and AML patient blasts, respectively]. SHIP1 protein levels were increased in all samples as well. SHIP1 is a tyrosine phosphatase that blocks PI3K-mediated membrane localization of AKT, which is often aberrantly activated in human cancers, including leukemia. Thus, we postulated that MLN4924-induced upregulation of SHIP1 via miR-155 downregulation would also result in PI3K/AKT pathway inhibition. As predicted, MLN4924 treatment of AML cell lines and primary blasts resulted in inhibition of the active AKT, as evidenced by a decline of phospho-AKTThr308 levels. Furthermore, the pharmacologic activity of MLN4924 was inhibited by forced expression of miR-155 in THP-1 cells and AML blasts, as shown by a partial loss of SHIP1 upregulation and caspase-3 activation, thus preventing MLN4924-triggered induction of apoptosis (p 〈 0.01) and decrease in cell viability (p 〈 0.05). In vivo, mature miR-155 levels in the peripheral blood of xenografted mice decreased by 50% after 24hrs and 80% after 48hrs (p 〈 0.01) from the first dose of MLN4924. Moreover, 21 days from the start of MLN4924 treatment, the average white blood cell count was significantly lower in the MLN4924-treated group (5,333 cells/ul ± 1040) compared with the vehicle-treated group (36,166 cells/ul ± 10,598; p 〈 0.01). The average spleen weight was also dramatically reduced in the MLN4924-treated group (58.06 mg ±12.74) compared with the control group (305.66 mg ±51.1; p 〈 0.01). Importantly, MLN4924 significantly prolonged the survival of leukemic mice; median survival was 45.5 vs. 31 days for MLN4924-treated vs. control groups (p 〈 0.0001, n=10 per group), respectively. Conclusions We showed that MLN4924 treatment of AML cells in vitro and in vivo resulted in decreased miR-155 expression, reactivation of its target gene, SHIP1, and concomitant inhibition of PI3K/AKT pathway. Our data also support that miR-155 downregulation is a critical component of MLN4924’s antileukemic activity. Thus, our work provides novel insight into MLN4924’s mechanism of action and the rationale for combining this drug with emerging anti-microRNA compounds. Disclosures: No relevant conflicts of interest to declare.

Abstract: The Acute Myeloid Leukemia (AML) subtype characterised by translocations of the Mixed-Lineage Leukemia gene, MLL (t11q23; MLL-AML), is a particularly devastating disease with a median overall survival of only 9 months with current standard therapy. Cyclin dependent kinase (CDK) 9 inhibitors (CDK9i) directly target the CDK9/cyclin T complex (pTEFb) that is essential for activity of the MLL-fusion proteins and for transcriptional elongation, and therefore leads to reduction of transcript levels for multiple key leukemic oncogenes e.g. HOXA9, MYC and MCL1. Several observations suggest that utilising CDK9i to simultaneously target these oncogenes will be an effective strategy for AML, and MLL-AML in particular: (i) Leukemic stem cell (LSC) fractions of AML cells express a high level of MCL1, (ii) Targeting MCL1 has been demonstrated to reduce leukaemia cell survival in a murine model of MLL-ENL, (iii) MCL1 is consistently elevated in AML patients at relapse, (iv) HOXA9 is critical for leukemogenesis in many AMLs, in particular MLL-AML, (v) MYC has been shown to be a critical oncogene in MLL-AML, and (vi) CDK9 function has been shown to be important for MYC-driven tumorigenesis. Our in vitro and in vivo data support the clinical potential of a novel orally bioavailable inhibitor of CDK9, CDKI-73, as an effective therapy for MLL-AML patients. CDKI-73 is a potent inhibitor of CDK9 (Ki 3.5nM)1 and has been shown to induce down-regulation of MCL1, and cell death of Chronic Lymphocytic Leukemia (CLL) B-cells2 and Ovarian Cancer (OvCa) cells3 with nanomolar potency. At doses that are highly toxic for tumour cells, CDKI-73 shows limited toxicity for normal T- and B- Lymphocytes, Bone Marrow Mononuclear cells (BMMNC) and normal colony forming cells (CFC) from the BMMNC fraction. CDKI-73 has many favorable properties also making it an excellent clinical candidate for AML when compared to other CDK9i; in particular, CDKI-73 is (i) unique in its spectrum of inhibition, including targeting CDK6 (IC50 = 0.038 µM; a critical kinase for MLL-AML4), and is (ii) orally bioavailable (F = 56%)2, facilitating sustained in vivo target inhibition. Here we present data showing that in MLL-AML cell lines, CDKI-73 induces growth suppression and apoptosis associated with rapid loss of Myc and MCL1, and activation of PARP. In primary AML patient samples treated with 200nM CDKI-73, we have observed a similar decrease in MCL1 protein levels, with increased 7AAD uptake and Annexin-V staining, consistent with apoptotic cell death. Using a subcutaneous MV4;11 nude mouse xenograft model, we have shown that oral dosing of CDKI-73 (100 mg/kg once every 3 days for 18 days) resulted in a high level of anti-tumour efficacy (p 〈 0.0001 compared to vehicle-treated mice), with minimal toxicity. Moreover, for an established MLL-AML patient-derived xenograft (PDX) generated in NOD/SCID-IL2RG-/- (NSG) mice we also observed significant inhibition of human AML in peripheral blood (p 〈 0.0001), BM (p 〈 0.05) and spleen (p 〈 0.001) with administration of CDKI-73 at 75 mg/kg every 3 days for 15 days. In both models CDKI-73 was well-tolerated at these doses, consistent with our published and preliminary data showing differential effects of CDKI-73 on tumour versus normal cell populations. Given this data, our priority now is to establish the effectiveness of CDKI-73 across a larger panel of primary MLL-AML samples, in further patient derived AML xenografts, and as a combination treatment with AML chemotherapy. REFERENCES: 1. Shao H, Shi S, et al. (2013). J Med Chem. 56(3):640-59. 2. Walsby E, Pratt G, et al. (2014). Oncotarget. 5(2):375-85. 3. Lam F, Abbas AY, et al. (2014). Oncotarget. 5(17):7691-704. 4. Placke T, Faber K, et al. (2014). Blood. 124(1):13-23. Disclosures No relevant conflicts of interest to declare.