Patient-derived Research Articles

Abstract 4030: Efficacy of novel combination therapy against AML cells with mutant NPM1

Abstract Cytoplasmic dislocation and loss of nucleolar localization of the chaperone protein NPM1c occurs in 30% of AML following a frame shift C-terminal mutation in exon 12 of NPM1 (creating NPM1c), which contributes to differentiation arrest, growth and survival of AML stem progenitor cells. In present studies, we determined that knock out (KO) of mutant (mt) NPM1 (NPM1c) in the AML OCI-AML3, utilizing targeted gRNA (compared to control) and CRISPR-Cas9, induced growth arrest, morphologic differentiation and loss of viability of the KO cells over 5 to 9 days. This was associated with depletion of protein levels of NPM1c, c-Myc, and MEIS1, but upregulation of PU.1, RUNX1, CD11b, p21 and NOXA levels. ChIP-Seq analysis with H3K27Ac antibody 5d after KO demonstrated reduced peak density at the super-enhancers of MYC, MEIS1, and HOXA9. RNA-Seq analysis showed negative enrichment of HALLMARK gene sets of mRNA expression of MEIS1/HOXA9, MYC targets, ribosome/translation and cell cycle. Compared to OCI-AML3 with NPM1c, OCI-AML3 with NPM1 KO exhibited abrogation of the Menin inhibitor SNDX-50469-induced differentiation and apoptosis. Additionally, NPM1 KO also significantly reduced sensitivity of OCI-AML3 cells to apoptosis induced by targeted agents previously shown to demonstrate efficacy against AML cells with NPM1c, including KPT-330 (XPO1 inhibitor), homoharringtonine (protein translation inhibitor), and anti-AML chemotherapeutic agents, including daunorubicin, cytarabine and etoposide. However, NPM1 KO sensitized OCI-AML3 cells to ATRA (all trans retinoic acid)-induced differentiation and loss of viability, which was associated with marked induction of p21 and CD11b. Utilizing RNA-Seq signature of NPM1c KO and interrogating the LINCS1000-CMap data set of gene expression signatures, we determined that among the top expression mimickers were several pan-HDAC inhibitors and a WEE1 kinase inhibitor. Treatment with adavosertib (WEE1 inhibitor, MK-1775) or romidepsin (HDAC inhibitor) dose-dependently induced apoptosis in OCI-AML3 cells, as well as in 5 samples of patient-derived (PD) AML cells with NPM1c. Moreover, co-treatment with adavosertib and romidepsin synergistically induced lethality in OCI-AML3 and PD AML cells with NPM1c. This was associated with marked reduction in protein expressions of c-Myc, c-Myb, MEIS1 and CDK4/6, but upregulation of TP53 and p21 levels. These findings highlight that presence of NPM1c mechanistically regulates the sensitivity of AML cells to Menin inhibitor, ATRA and anti-AML chemotherapeutic agents. They also identify the novel non-chemotherapy combination of pan-HDAC inhibitor and WEE1 inhibitor for its potential synergistic activity against AML with NPM1c. Citation Format: Christopher P. Mill, Warren C. Fiskus, John A. Davis, Courtney D. DiNardo, Christine Birdwell, Qi Jin, Koichi Takahashi, Joseph D. Khoury, Kapil N. Bhalla. Efficacy of novel combination therapy against AML cells with mutant NPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4030.

Progenitor cells derived from gene-engineered human induced pluripotent stem cells as synthetic cancer cell alternatives for in vitro pharmacology.

Limitations in genetic stability and recapitulating accurate physiological disease properties challenge the utility of patient-derived (PD) cancer models for reproducible and translational research. A portfolio of isogenic human induced pluripotent stem cells (hiPSCs) with different pan-cancer relevant oncoprotein signatures followed by differentiation into lineage-committed progenitor cells was genetically engineered. Characterization on molecular and biological level validated successful stable genetic alterations in pluripotency state as well as upon differentiation to prove the functionality of our approach. Meanwhile proposing core molecular networks possibly involved in early dysregulation of stem cell homeostasis, the application of our cell systems in comparative substance testing indicates the potential for cancer research such as identification of augmented therapy resistance of stem cells in response to activation of distinct oncogenic signatures.

MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines.

Simple SummarySince 2005, temozolomide (TMZ) has been used as a standard first-line treatment for glioblastoma (GBM, grade IV glioma), and despite many studies and efforts no better alternatives have emerged. Tumor recurrences and TMZ resistance are common and the prognosis is very poor with a median overall survival of 14–16 months. The development of new pharmacological strategies is even more difficult due to the presence of glioma stem cells (GSCs). In this multidisciplinary study, we tested the imidazobenzoxazin-5-thione MV1035, alone and in combination with TMZ, in U87-MG and patient-derived (PD) GSCs in order to demonstrate a putative synergic effect. MV1035 was tested following its in silico predicted ability to act as an inhibitor against ALKBH2 and ALKBH5, both involved in maintaining the tumorigenicity of glioblastoma.Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Pre-Clinical Efficacy of Targeting TBL1/R-β-Catenin-TCF7L2 Axis Against AML with 3q26 Lesions and EVI1 Overexpression

EVI1 gene maps to the MECOM locus at chromosome 3q26.2 and encodes for a zinc finger domain-containing transcriptional regulator. EVI1 supports hematopoietic stem cell self-renewal and blocks hematopoietic differentiation. EVI1 is overexpressed in up to 10% of AML, including those harboring chromosome translocation t(3;3)(q21;q26.2) or inv(3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression due to 3q26.2 lesions in MDS and AML is frequently associated with monosomy 7 and confers poor response to therapy and inferior relapse-free and overall survival. We had previously reported the pre-clinical efficacy of targeting TBL1/R1-nuclear β-catenin-TCF7L2 by tegavivint (BC-2059, Iterion Therapeutics) against AML stem/progenitor cells. This led us to interrogate the anti-AML activity of tegavivint (TV) in AML models harboring 3q26.2 lesions, where EVI1 overexpression has been documented to drive the biology of AML stem/progenitor cells. For this, we utilized AML cell lines with 3q26.2 lesions with/without monosomy 7 (UCSD-AML1, OCI-AML20, AML191 / MUTZ-3, AML194, HNT34), as well as patient-derived (PD) AML cells with 3q26 lesions with or without monosomy 7. Treatment with TV (10 to 100 nM) dose-dependently induced apoptosis in these cellular models. This was associated with attenuation of protein levels (determined by immunoblot analyses) of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, pan-BET protein inhibitor OTX015 (100 to 1000 nM) dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). Following TV treatment, RNA-Seq and gene set enrichment analysis in UCSD-AML1 and OCI-AML20 cells showed log2 fold-changes in gene expression and positive enrichment of pathway genes and/or reactomes of inflammatory response, TNFα and interferon signaling, TGFβ, NOTCH and apoptosis signaling, as well as negative enrichment of gene sets of c-Myc, E2F, G2M checkpoint, DNA replication and repair and chromosome maintenance (all with FDR q-values < 0.1). QPCR analysis showed repression of EVI1, MYC and KIT, but upregulation of Axin2 mRNAs. Following TV treatment, confocal microscopy showed reduction of nuclear protein levels of EVI1 and β-catenin, as well as disrupted their co-localization with TBL1. Proximity ligation assay also demonstrated that exposure to TV significantly reduced the proximity of TBL1 and EVI1 as well as of TBL1 and β-catenin. Mass cytometry (CyTOF) analysis of patient-derived (PD) AML samples with t(3:3)/inv(3) confirmed that TV treatment attenuated protein levels of EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67, but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). Consistent with effects of TV on gene-expressions that regulate cell death pathways, in vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (as determined by the SynergyFinder algorithm) of AML cell lines and PD AML cells with t(3:3)/inv(3) and EVI1 overexpression. In the in vivo HNT-34 model of flank-implanted and established tumors in athymic nude mice, treatment with TV (50 mg/kg, TIW, IP) and venetoclax (30 mg/kg, PO, daily) for 5-weeks yielded more tumor growth delay than vehicle control or TV alone. Additionally, following tail vein infusion and engraftment of luciferized AML191 cells in NSG mice, treatment with TV and/or venetoclax or OTX015 (30 mg/kg, PO, daily) was evaluated for 6 weeks. Co-treatment with TV and venetoclax or TV and OTX015 significantly reduced AML growth (determined by reduction in bioluminescence by Xenogen camera) (p < 0.05), as well as improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. Collectively, these findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 by treatment with TV also inhibits EVI1 and its targets. They also demonstrate superior pre-clinical efficacy of novel TV-based combinations with BCL2 or BET protein inhibitor against AML models harboring 3q26 lesion and EVI1 overexpression. DisclosuresDiNardo: Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy. Horrigan: Iterion Therapeutics: Current Employment. Kadia: Astellas: Other; Cure: Speakers Bureau; Genfleet: Other; Ascentage: Other; Jazz: Consultancy; Sanofi-Aventis: Consultancy; Dalichi Sankyo: Consultancy; Cellonkos: Other; Novartis: Consultancy; AstraZeneca: Other; BMS: Other: Grant/research support; Liberum: Consultancy; Amgen: Other: Grant/research support; Pulmotech: Other; Pfizer: Consultancy, Other; Genentech: Consultancy, Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support.

Preclinically Effective Menin Inhibitor SNDX-50469 and SNDX-5613-Based Combinations Against MLL1-Rearranged (MLL-r) or NPM1-Mutant AML Models

Preclinically Effective Menin Inhibitor SNDX-50469 and SNDX-5613-Based Combinations Against MLL1-Rearranged (MLL-r) or NPM1-Mutant AML Models

Clinical-Stage Menin Inhibitor KO-539 Is Synergistically Active with Multiple Classes of Targeted Agents in KMT2A-r and NPM1-Mutant AML Models

The N-terminus of the histone-lysine-N-methyltransferase MLL1 (KMT2A) contains the menin binding domain (MBD). Menin is a scaffold protein that binds to MBD and tethers MLL1 to chromatin. The Menin-KMT2A complex regulates expression of the leukemogenic homeobox A9 (HOXA9) gene and its co-factor MEIS1 in myeloid stem progenitor cells. In MLL-rearranged (MLL-r) AML, the N-terminus of the KMT2A gene is fused to the C-terminus of any of over 80 fusion partners, most commonly AF4, AF9, ENL and ELL. These MLL fusion partners are components of (and recruit) the super elongation complex and DOT1L to induce H3K4Me3 and H3K79Me2 marks on active chromatin, driving aberrant expression of HOXA9, MEIS1, PBX3, MEF2C and CDK6. In AML with mutant NPM1 (NPM1c), the wild type menin-KMT2A complex is the main oncogenic driver of HOXA9, MEIS1 and FLT3, promoting self-renewal of mutant leukemic blasts. KO-539 is a novel, oral investigational drug candidate targeting the Menin-KMT2A protein-protein interaction. Preclinical data show that KO-539 and close analogs induce differentiation and loss of viability of AML cells, and exert profound anti-leukemic activity in multiple PDX models harboring MLL-FP or NPM1c when dosed continuously QD for 3-6 weeks. In an ongoing phase 1/2A KOMET-001 clinical trial (NCT04067336) evaluating KO-539 in adult patients with relapsed and/or refractory AML, significant biological activity as well as complete remission lacking minimal residual disease have been observed.The present studies were focused on further elucidating biologic effects and on determining synergistic activity of KO-539 with agents targeting BCL2, BET proteins and CDK6. Treatment with KO-539 dose-dependently inhibited in vitro growth, as well as induced differentiation (by both morphology and increased CD11b expression) and loss of viability of MOLM13 (MLL-AF9 and FLT3-ITD) and OCI-AML3 (NPM1c and homozygous NRAS mutation) cells and patient-derived (PD) MLL-r or NPM1c AML cells. This was associated with repression of MEIS1, PBX3, MEF2C, FLT3, MYC, BCL2 and CDK6 transcription but increased mRNA expression of ITGAM (CD11b). KO-539 treatment also dose-dependently depleted protein levels of Menin, MEIS1, FLT3, CDK6 and BCL2, but upregulated MCL1 and CD11b proteins in MOLM13 and OCI-AML3 cells. Mass cytometry (CyTOF) analysis of PD MLL-r and NPM1c AML samples confirmed that, following KO-539 treatment there was also decline in protein levels of Menin, MEIS1, MEF2C, PBX3, FLT3, CDK6 and BCL2 in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). KO-539-mediated Menin depletion was associated with polyubiquitylation and proteasomal degradation of Menin protein, given that protein levels were restored by co-treatment with the proteasome inhibitor carfilzomib. Notably, co-treatment (in vitro) with KO-539 in combination with venetoclax, OTX015 (pan-BET inhibitor) or abemaciclib (CDK6 inhibitor) for 72 to 96 hours induced synergistic loss of viability in cultured cell lines and PD AML cells from both MLL-r and NPM1c AML but not normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c, as determined by the SynergyFinder algorithm. Findings of ongoing in vivo studies determining effects of treatment with KO-539 and/or venetoclax or OTX015, versus vehicle control in PDX models will be presented at the meeting. These preclinical findings highlight the molecular correlates of anti-AML efficacy of KO-539 and demonstrate potentially synergistic KO-539-based combinations with inhibitors of BCL2, BET proteins and CDK6 against MLL-r or NPM1 mutant AML. DisclosuresDaver: Bristol Myers Squibb: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Genentech: Consultancy, Research Funding; Glycomimetics: Research Funding; Amgen: Consultancy, Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Kadia: Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support; Amgen: Other: Grant/research support; BMS: Other: Grant/research support; Cure: Speakers Bureau; Genentech: Consultancy, Other: Grant/research support; Dalichi Sankyo: Consultancy; AstraZeneca: Other; Astellas: Other; Genfleet: Other; Ascentage: Other; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other; Jazz: Consultancy; Liberum: Consultancy; Novartis: Consultancy; Pfizer: Consultancy, Other. DiNardo: Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Leoni: Kura Oncology: Current Employment. Burrows: Kura Oncology: Current Employment.

Homoharringtonine and Mebendazole Are Preferentially Lethal Against Models of Myeloid Malignancy Associated with Germline Mutant RUNX1

RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Majority of mono-allelic germline mutations in RUNX1 are missense, large deletions or truncation mutations, behaving mostly as loss of function (LOF) mutations. They are ~40%-penetrant and cause Familial Platelet Disorder (RUNX1-FPD) that has a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. FPD-MM harbors co-mutations, most commonly on the second allele of RUNX1, and on BCOR, PHF6, K-RAS, WT1 or TET2, which confer relative resistance to standard therapy for MDS or AML. Although curative in some patients with FPD-MM, allogeneic transplantation from matched, un-related donors carries risk of graft versus host disease and frequent AML relapse. This creates a strong rationale and an unmet need to develop novel targeted therapies for FPD-MM. We previously reported on utilizing the RNA-Seq signature of RUNX1 knockdown, which exerted more lethality in AML cells with mutant (mt) RUNX1 compared to AML harboring two copies of wild-type RUNX1, for conducting LINCS1000-CMap analysis. This identified several expression mimickers (EMs), including the protein synthesis inhibitor homoharringtonine (HHT or omacetaxine) and anthelmintic fenbendazole (analog of mebendazole). Present studies demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in four patient-derived (PD) bone marrow aspirate (BMA) samples of FPD-MM (3 AML and 1 MDS) compared to RUNX1-FPD (3 samples) or in normal CD34+ progenitor cells. In a patient with RUNX1-FPD (expressing mtRUNX1 K194N), who developed FPD-MM, following co-mutations were documented by NGS: BCOR A1437fs, PHF6 L324fs, SF3B1 D781G and SRSF2 P95R/L. From BMA of this patient, we successfully established the first ever, continuously cultured cell line (GMR-AML1) expressing the same germline mtRUNX1. GMR-AML1 cells were cytogenetically diploid and lacked MYC or MLL1 rearrangement, or any copy number gains or losses on array CGH. However whole exome sequencing (WES) identified additional mutations in TP53 (P72R), AIM2 (K340fs), NELFB (L523F), CEP152 (Y370X), SUGP2 (H23L), RRM2B (R71fs), TADA3 (T27R), SPDYE6 (G292C) and PRDM9 (S814R) with % VAF ranging between 33 to 55%. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL3R), CD86 and CD33, but without expression of CD34, CD14, CD11b, MPO or CD135 (FLT3). Compared to the AML OCI-AML5 cells with somatic mtRUNX1, GMR-AML1 cells demonstrated markedly reduced protein expression of RUNX1, RUNX2, PU.1, c-Myb, GFI1, GFI1B, FLT3, MEIS1 and CEBPα (p42), but much higher protein expression of RUNX3 and NOTCH (p120). CRISPR-Cas9 knockout of RUNX3 in GMR-AML1 cells restored RUNX1 expression, while significantly increasing % of differentiated cells. Although dose-dependently sensitive to daunorubicin, etoposide, cytarabine and panobinostat (class I and II HDAC inhibitor), GMR-AML1 cells were relatively insensitive to venetoclax, A1155463 (Bcl-xL inhibitor), AZD-5991 (MCL1 inhibitor), azacytidine or decitabine. Notably, treatment with HHT or MB dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 40 and 330 nM, respectively). Additionally, co-treatment with HHT and venetoclax synergistically induced apoptosis in GMR-AML1 cells, as determined by the SynergyFinder algorithm. This synergy in GMR-AML1 cells was associated with abrogation of venetoclax-induced increase in MCL1 and Bcl-xL levels, as well as greater decline in levels of RUNX3, PU.1, c-Myb, c-Myc, MPL and CDK4/6. Tail vein infusion and engraftment of luciferase-transduced GMR-AML1 (10 6 cells) caused marked splenomegaly and 100% mortality of NSG mice by day-18, post-infusion. We will present at the ASH meeting findings of ongoing in vivo studies determining effects of treatment with HHT and/or venetoclax, versus vehicle control, on AML burden and overall survival of NSG mice engrafted with GMR-AML1 cells. Overall, preclinical findings presented here highlight the molecular and genetic features associated with progression of RUNX1-FPD to FPD-MM, especially in the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are preferentially more lethal against FPD-MM versus RUNX1-FPD cells and exert synergistic lethality with venetoclax against GMR-AML1 cells. DisclosuresDiNardo: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; ImmuneOnc: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Takahashi: GSK: Consultancy; Celgene/BMS: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Khoury: Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

Targeted Therapy for AML Expressing RUNX1 Mutation

RUNX1 is the DNA-binding subunit of the core binding factor (CBF) complex and a transcription factor (TF) involved in normal and malignant hematopoiesis. Transcriptionally active full-length RUNX1b isoform is transcribed from 6 exons, encoding a DNA binding ‘Runt’ homology domain and a more C-terminal transcription activation domain. Within its CTCF-anchored loop or TAD (topologically-associating domain), RUNX1 gene has a highly conserved +24-kb enhancer (eR1 in mouse), which is part of a large super-enhancer (>170 kb) covering the entire intron 1 between proximal P2 and distal P1 promoter of RUNX1 and controlling the transcription of RUNX1. TFs bind to super-enhancers/enhancers that loop over to interact with promoters to transcriptionally regulate oncogenes, such as RUNX1 and its targets, as well as multiple other AML-relevant oncogenes involved in growth and survival of AML blast progenitor cells (BPCs). Heatmap of Hi-C interaction scores within the RUNX1 TAD showed triangle shaped regions of high interaction scores highlighting interactions between +24-kb enhancer and the P2 promoter of RUNX1. To functionally validate these findings, we cloned and transfected into HEK293 cells expressing RUNX1, a luciferase reporter construct containing the DNA of the P2 promoter with or without the +24 kb-enhancer of RUNX1. Our findings confirm that, as compared to the promoter or the enhancer alone, the +24 kb-enhancer-driven P2 promoter exhibited superior luciferase induction representing increased transcriptional activity of RUNX1. Somatic mutations in RUNX1 commonly occur in MDS, as well as in the secondary (s) or de novo AML (~10%). Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function mutations. Presence of mtRUNX1 confers relative therapy-resistance and poorer survival in patients with AML. Lack of specific targeted therapy coupled with resistance to standard chemotherapy may account for the poorer outcome in AML expressing somatic or germline mtRUNX1. Therefore, there is a strong rationale and an unmet need to develop novel targeted therapies for AML expressing somatic or germline mtRUNX1. In the present studies, we demonstrate for the first time that shRNA mediated knockdown of RUNX1 expression inhibits growth and induces differentiation and apoptosis of cultured cell line (OCI-AML5) and patient-derived (PD) AML BPCs expressing mtRUNX1. After engraftment, doxycycline-inducible shRNA-mediated in vivo knockdown of RUNX1 in OCI-AML5 cells, compared to controls, markedly reduced AML burden and improved survival of the immune-depleted (NSG) mice engrafted with luciferase transduced OCI-AML5 cells. Interaction of TFs with DNA and transcriptional co-factors such as the histone acetyltransferase p300 at enhancers and promoters leads to acetylation of histone proteins and TFs. BET (Bromodomain Extra-terminal) protein (BETP) BRD4 binds to acetylated histones and TFs at super-enhancers and recruits transcriptional co-factors to promote RNA pol II (RNAP2)-mediated transcription of oncogenes and their targets. ChIP-Seq analysis with anti-BRD4 and anti-H3K27Ac antibody showed that Runx1 super-enhancer and P2 promoter are occupied by BRD4. shRNA mediated knockdown of BRD4 attenuated expression of RUNX1 and its targets, MYC, PU.1 and myeloperoxidase (MPO), inhibited growth and induced apoptosis of OCI-AML5 cells. We also found that BETP-PROTACs (proteolysis targeted chimera) ARV-825 and its pharmacologically superior version ARV-771 (20 to 250 nM) caused degradation and depletion of BRD4, reduced BRD4 occupancy at the+24-kb-enhancer and P2 promoter of RUNX1, repressing RUNX1 and its targets, and potently induced apoptosis of OCI-AML5, Mono-Mac-1 and PD AML BPCs expressing mtRUNX1. Treatment with ARV-771 (30 mg/kg SQ daily for 3 weeks also inhibited AML growth and improved survival of NSG mice engrafted with luciferase-transduced OCI-AML5 cells. By utilizing the RNA-Seq mRNA signature from RUNX1-depleted AML cells we queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. This uncovered several novel agents that inhibit RUNX1 expression and induce in vitro lethality and reduce in vivo leukemia burden in AML BPCs expressing mtRUNX1. These findings highlight RUNX1-targeted novel agents to be developed and tested as potential therapy for AML expressing mtRUNX1. DisclosuresDiNardo:AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Qian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Takahashi:Symbio Pharmaceuticals: Consultancy.

A brain-penetrant microtubule-targeting agent that disrupts hallmarks of glioma tumorigenesis.

BackgroundGlioma is sensitive to microtubule-targeting agents (MTAs), but most MTAs do not cross the blood brain barrier (BBB). To address this limitation, we developed the new chemical entity, ST-401, a brain-penetrant MTA.MethodsSynthesis of ST-401. Measures of MT assembly and dynamics. Cell proliferation and viability of patient-derived (PD) glioma in culture. Measure of tumor microtube (TM) parameters using immunofluorescence analysis and machine learning-based workflow. Pharmacokinetics (PK) and experimental toxicity in mice. In vivo antitumor activity in the RCAS/tv-a PDGFB-driven glioma (PDGFB-glioma) mouse model.ResultsWe discovered that ST-401 disrupts microtubule (MT) function through gentle and reverisible reduction in MT assembly that triggers mitotic delay and cell death in interphase. ST-401 inhibits the formation of TMs, MT-rich structures that connect glioma to a network that promotes resistance to DNA damage. PK analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft flank tumor mouse model demonstrates significant antitumor activity and no over toxicity in mice. In the PDGFB-glioma mouse model, ST-401 enhances the therapeutic efficacies of temozolomide (TMZ) and radiation therapy (RT).ConclusionOur study identifies hallmarks of glioma tumorigenesis that are sensitive to MTAs and reports ST-401 as a promising chemical scaffold to develop brain-penetrant MTAs.

EXTH-53. A BRAIN-PENETRANT MICROTUBULE-TARGETING AGENT THAT DISRUPTS HALLMARKS OF GLIOMA TUMORIGENESIS

Abstract Most cancer therapeutics developed to date are excluded from the brain and are therefore ineffective in treating glioma, the most devastating type of brain cancer in adults. Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of solid and hematopoietic tumors, and evidence suggests that glioma is sensitive to MTAs; but most MTAs do not cross the blood-brain-barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioma in culture at nanomolar concentrations. ST-401 bound to the colchicine site, inhibited tubulin assembly and reversibly reduced microtubule (MT) dynamics. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity does not correlate with any of the compounds tested thus far through this platform but showed weak correlations for two MTA that work through distinct mechanisms: taxol (p=0.46) and vinblastine (p=0.34). Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function. We discovered that ST-401 killed patient-derived (PD) glioma isolates in both mitosis and interphase, and inhibited the formation of tumor microtubes, MT-rich structures that connects glioma cells to a network that is resistant to standard therapies. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft model demonstrated significant antitumor activity. In an immunocompetent mouse model of platelet-derived growth factor B-driven glioma, ST-401 significantly enhanced the therapeutic efficacies of standard care therapies temozolomide and radiation therapy. Our study identified novel aspects of glioma tumorigenesis that exhibit enhanced sensitivity to MTAs and shows that the brain-penetrant MTA, ST-401, represents a promising chemical scaffold to develop therapeutics for the treatment of patients diagnosed with glioma.

Abstract 3913: Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models

Abstract Cancer organoids are heterogeneous 3D cellular clusters with complexities that mimic some characteristics of tumors in situ. Thus, assays performed with cancer organoids might enable better predictions of in vivo drug responses than those performed with cell monolayers. The National Cancer Institute (NCI) is developing a national repository of Patient-Derived (PD) models comprised of clinically annotated and molecularly characterized PD xenografts (PDXs), PD tumor cell lines (PDCs), and PD cancer organoids (PDOrgs) (https://pdmr.cancer.gov/). We evaluated the therapeutic activity of a panel of FDA-approved and investigational anticancer agents, including carboplatin, gemcitabine, paclitaxel, SN38, 5-FU, adavosertib, erlotinib, trametinib, and vemurafenib, against a cohort of PDCs, PDOrgs, and PDXs from solid tumors including colon, gastroesophageal, head and neck, NSCLC, pancreatic, bladder, and uterine cancers. Our goal was to investigate whether drug sensitivities determined using PDCs and PDOrgs correlate with responses observed in the matching PDXs. Cultures were exposed to anticancer agents at concentrations ranging from 1 pM to 100 µM for periods of 4 or 6 days. The data indicated that the GI50 values for PDOrgs were in overall agreement with in vivo PDX drug responses measured as relative median to event free survival (RMEFS), where an event is the median time (days) from treatment initiation to tumor volume quadrupling, calculated as median time to tumor volume quadrupling for treated animals/median time to tumor volume quadrupling for control animals. For both paclitaxel and trametinib, responses in PDOrgs, from most sensitive to most resistant, were similar to the corresponding PDXs. Drug sensitivities determined in PDC monolayers were less clearly related to in vivo PDX responses; particularly for PDCs treated with carboplatin, gemcitabine, and SN-38. This work is part of a larger effort to provide a rigorous comparison between fully characterized and annotated PDCs-PDOrgs-PDXs to assess the value of different in vitro model systems for the prediction of PDX drug responses. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Petreena Campbell, Curtis Hose, Lara El Touny, Erik Harris, John Connelly, Carrie Bonomi, Kelly Dougherty, Savanna Styers, Abigail Walke, Jenna Moyer, Mariah Baldwin, Anna Wade, Michael Mullendore, Kaitlyn Arthur, Matthew Murphy, Kevin Plater, Marion Gibson, Joseph Geraghty, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Tiffanie Chase, John Carter, Howard Stotler, Debbie Trail, Luke Stockwin, Dianne Newton, Yvonne Evrard, Melinda Hollingshead, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, James H. Doroshow, Annamaria Rapisarda. Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3913.

Abstract GMM-023: PATIENT DERIVED XENOGRAFTS (PDXS) AND PDX DERIVED TUMOR CELLS (PDTC) ALLOW THE IDENTIFICATION OF ACTIONABLE CANCER GENES AND TREATMENT OPTIONS FOR PLATINUM REFRACTORY/RESISTANT OVARIAN CARCINOMAS

Abstract Patients with advanced ovarian cancers have experienced little improvement in overall survival with standard treatments even after the incorporation of anti-angiogenic therapies. Besides anti-PARP inhibitors, matching individual critical genomic alterations with the best available drugs has not advanced as in other cancers, likely because a handful of cancer-related genes are mutated at high frequency, while many more are found mutated at much lower frequencies. This so called “mutation tail” is not only long but also mostly unexplored. We used Patient Derived Xenografts (PDXs) to identify actionable cancer genes and PDX Derived Tumor Cells (PDTCs) to accelerate the discovery of treatment options. We envisioned that the alleged weakness of PDX models, i.e. lack of human stromal and immune cells, might be instrumental to identify mutations in cancer and to test approved or experimental targeted drugs as monotherapy or in different combinations to link biomarkers to treatments. Forty-nine PDX lines from metastatic epithelial ovarian carcinomas have been propagated and fully characterized as far as histology, immunohistochemistry of epithelial and high-grade serous-specific markers and presence of TP53 and BRCA1/2 mutations. Copy number variations (CNV) analysis and Whole Exome Sequencing (WES) were carried out PDX lines derived from naïve metastatic high-grade epithelial ovarian carcinomas, which came out to be refractory/resistant to platinum drugs. We studied non-synonymous mutations with allele frequencies ≥0.1. Only mutations in cancer genes listed in databases were further analyzed. SNPdb allowed ruling out polymorphisms. SIFT and PROVEAN softwares predicted deleterious or damaging effects onto the protein sequences. DGIdb helped selecting actionable genes. We identified in one PDX line, a possibly loss-of-function mutation of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) had an allele frequency=0.9 in early and late passages. Moreover, in two micro-dissected FFPE samples of the source tumor this mutation had an allele frequency nearly identical to that of the mutated TP53. Hence, PIK3R1W624R could be a trunk mutation in the PDX line and possibly in the human counterpart. Treatment options were assayed ex-vivo, on short-term cultures of PDTCs of the PIK3R1W624R PDX line. Buparlisib, a pan-class I PI3K inhibitor, showed the ability to block proliferation of PDTCs and the growth in vivo of PDXs in regression preclinical trial. These data proofed-the-concept that a PDX-based pipeline is able to unveil actionable pathways for the treatment of advanced/metastatic ovarian cancer. Citation Format: Martina Olivero, Jessica Erriquez, Maddalena Arigoni, Sonia Capellero, Concetta D'Ambrosio, Gloria Mittica, Fulvio Borella, Dionyssios Katsaros, Silvana Privitera, Enrico Berrino, Tiziana Venesio, Giorgio Valabrega, Raffaele Calogero and Maria Flavia Di Renzo. PATIENT DERIVED XENOGRAFTS (PDXS) AND PDX DERIVED TUMOR CELLS (PDTC) ALLOW THE IDENTIFICATION OF ACTIONABLE CANCER GENES AND TREATMENT OPTIONS FOR PLATINUM REFRACTORY/RESISTANT OVARIAN CARCINOMAS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-023.

Abstract 4835: UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers

Abstract Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer activities of UD-017 in vitro and in vivo using patient-derived (PD) cancer cells. Methods: We first evaluated an antiproliferative activity of UD-017 broadly in a panel of cancer cell assay including patient-derived 3-dimensional tumor clonogenic assay. To verify the potential of c-Myc expression as a biomarker, we investigated the correlation between c-Myc expression levels and anticancer activity in vitro and in vivo using a colorectal cancer cell. In the main part, anticancer efficacy of UD-017 was investigated in vivo in PD-xenograft (PDX) models using representative PD cancer cells. Results: In antiproliferative panel assays using over 100 cancer cell lines, UD-017 broadly inhibited a wide range of cancer cells from colon, breast, lung, kidney, blood, pancreas, osteosarcoma, sarcoma and urinary bladder cancers. Especially, UD-017 showed high sensitivity in solid tumor such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, osteosarcoma and sarcoma cells with IC50s of 10-200 nM. UD-017 showed a good correlation of c-Myc expression levels with antiproliferative activity using colorectal cancer cells in vitro, and UD-017 reduced the intratumoral MYC mRNA levels by an administration at 100 mg/kg in a HCT-116 xenograft model in mice. In PDX models, UD-017 showed strong inhibition and even regression of tumor growth, and the tumors were almost disappeared on day 14 without body weight loss in non-small cell lung cancer (LXFL1121). In a pleuramesothelioma (PXF541) PDX model, UD-017 also showed regressive effect, and in gastric cancer (GXA3067) and sarcoma (SXFS117) PDX models, UD-017 completely inhibited the tumor growth. Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete antitumor responses in patient-derived xenograft models of diverse cancer types. c-Myc expression in cancer cells may be a biomarker for the antitumor effect of UD-017. These data support the rationale for further advancing towards clinical development. Citation Format: Takashi Matsushita, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Ayumi Ogawa, Toru Hasegawa, Yasunori Tokunaga, Yasuhiro Aga, Shigeru Ushiyama. UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers [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 4835.

Abstract 5067: BET protein proteolysis targeting chimera (BETP-PROTACs) exert more potent activity than BETP bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells

Abstract In BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 &amp; 2 inhibitor (JAKi), which is effective in the therapy of MPN-MF but does not significantly impact the clinical outcome in post-MPN sAML. We have previously reported that treatment with BETi, e.g. JQ1 or OTX015 inhibits growth and induces apoptosis of cultured sAML cells, including those that express JAK2 V617F and mutant TP53, e.g. HEL92.1.7 and SET2, as well as patient-derived (PD) CD34+ sAML cells. BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. However, treatment with BETi leads to the accumulation of BETP, e.g. BRD4, which may reduce BETi-mediated repression of c-MYC, NFκB and BETP-regulated oncoproteins. In contrast, BETP-PROTACs (proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.) degrade BETPs (including BRD4) in the cultured and PD CD34+ sAML cells. At equimolar concentrations, BETP-PROTACs were significantly more potent than the BETi in inducing apoptosis of cultured and PD sAML cells (p &amp;lt; 0.05), while sparing the CD34+ normal hematopoietic progenitor cells. Notably, BETP-PROTACs caused efficient and prolonged depletion of the levels of BETPs, including BRD4 (&amp;gt; 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, as determined by RNA-Seq and reversed phase protein array (RPPA) analyses, respectively. As compared to treatment with BETi, BETP-PROTAC caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry also showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-Rb, while inducing p21 levels in the CD34+ sAML stem/progenitor cells expressing CD90, CD244, CD123 and TIM3-Fc. Compared to treatment with each agent alone, co-treatment with BETP-PROTAC and R was synergistically lethal against the cultured and PD CD34+ sAML cells. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 was synergistically lethal against R-sensitive and R-resistant sAML cells. As compared to treatment with vehicle control, R treatment alone, treatment with BETP-PROTAC ARV-771 alone or in combination with R significantly reduced the in vivo sAML burden and improved the median survival of the immune-depleted mice engrafted with luciferase-transduced HEL92.1.7 cells. These findings strongly support further in vivo development of the novel BETP-PROTACs-based combinations against post-MPN sAML. Note: This abstract was not presented at the meeting. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Kanak Raina, Taghi Manshouri, Kevin G. Coleman, Yimin Qian, Andrew P. Crew, Angela Shen, Christopher P. Mill, Baohua Sun, Misun Kim, Agnieszka J. Nowak, Srdan Verstovsek, Craig M. Crews, Kapil N. Bhalla. BET protein proteolysis targeting chimera (BETP-PROTACs) exert more potent activity than BETP bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2017-5067

Abstract LB-081: Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation

Abstract RUNX1 is a transcription factor involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 (mtRUNX1) have been documented in de novo and secondary AML (10%), MDS (~10%) and CMML (up to 37%). Germ-line, mono-allelic mutations and deletions in RUNX1 cause the highly-penetrant (~40%) autosomal dominant, Familial Platelet Disorder with a propensity to Myeloid Malignancy (FPD-MM). The majority of mtRUNX1 are missense mutations, large deletions or truncation-mutations in the DNA-binding ‘RUNT’ homology domain, or in the C-terminal transactivation domain. These mutations behave mostly as loss of function mutations, associated with relative resistance to standard chemotherapy and an unfavorable prognosis in AML. This highlights an unmet need to develop and test novel targeted therapies for AML due to germ-line or somatic mtRUNX1. In the present studies, we demonstrate that shRNA-mediated knockdown of mutant and wild-type RUNX1 repressed its target genes MYC, PU.1 and MPO (myeloperoxidase), as well as inhibited growth and induced apoptosis of AML cells expressing mtRUNX1 (OCI-AML5 and Mono-Mac-1). Ex vivo depletion of RUNX1 abrogated the leukemia initiating potential of OCI-AML5 cells. After engraftment, tetracycline-inducible shRNA-mediated in vivo knockdown of RUNX1, as compared to the non-induced controls, enhanced survival of the immune-depleted (NSG) mice engrafted with OCI-AML5 cells. RUNX1 transcription is driven by the BET protein BRD4-occupied super enhancer in the first intron of the RUNX1 gene. A heat map of Hi-C interaction scores within the RUNX1 TAD (topology-associated domain) showed triangle-shaped regions of high interaction, with CTCF binding sites defining the TAD boundaries, anchoring a loop that separates the RUNX1 TAD from other TADs. Consistent with this, our findings show that, shRNA-mediated depletion of BRD4 or treatment with BET protein (BETP) inhibitor (BETi) OTX015 reduced BRD4 occupancy at the enhancer and promoter of RUNX1. This was associated with depletion of RUNX1 and its target-gene expressions and apoptosis of cultured and patient-derived (PD), primary AML blast progenitor cells (BPCs). Additionally, treatment of NSG mice engrafted with luciferase-transduced OCI-AML5 cells with OTX015 (50 mg/kg/day X 5 days, for 3 weeks) reduced the AML burden and significantly improved their survival (p &amp;lt; 0.01). Co-treatment with the BETi and BCL2-antagonist venetoclax or CDK4/6 antagonist palbociclib or decitabine synergistically induced apoptosis of OCI-AML5 and PD AML BPCs (combination index values of &amp;lt; 1.0). Results describing the in vivo efficacy of BETi and BETP-PROTAC (proteolysis targeting chimera), alone and in combinations against AML expressing mtRUNX1 will be updated at the AACR meetings. Collectively, these findings highlight novel and effective, mechanistically-targeted, single agent or combination, BETP antagonist-based therapy of AML expressing mtRUNX1. Citation Format: Christopher P. Mill, Courtney DiNardo, Warren C. Fiskus, Dyana T. Saenz, Baohua Sun, Agnieszka J. Nowak, Misun Kim, Mark Routbort, Koichi Takahashi, Kapil N. Bhalla. Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-081. doi:10.1158/1538-7445.AM2017-LB-081

Abstract AP19: SINGLE–CELL RNA–SEQUENCING OF PATIENT–DERIVED OVARIAN CANCER CELLS AND PATIENT–DERIVED XENOGRAFT MODELS

Abstract BACKGROUND AND PURPOSE: Genetic heterogeneity is a hallmark of ovarian cancer (OvCa) biology and underlies treatment resistance. Macroscopic and or treatment resistant microscopic residual disease (MRD) after debulking surgery and chemotherapy are the source for disease recurrence and death in these patients. Current profiling methods are unable adequately reflect heterogeneity, and transcriptional programs associated with treatment resistance and MRD remain elusive. To elucidate transcriptional heterogeneity and potential mechanisms of drug-resistance, we isolated OvCa cells from patients with malignant ascites using flow-cytometry. We applied single-cell RNA-sequencing (sc-RNA-seq) to ascites-derived cells from patients with OvCa. We picked OvCa spheroids and profiled these separately. To investigate MRD, we used three PDX-models stably expressing mCherry, treated with carboplatin, and harvested tumor cells for sc-RNA-seq at three time points (pre-treatment, at time of MRD as determined by bio-luminescence imaging, and disease relapse). SUMMARY OF RESULTS: We successfully sequenced 770 single-cell transcriptomes from 6 individuals with treatment-resistant OvCa, including 3 patients with sequential samples. We mapped the landscape of chromosomal aberrations by inferred large-scale copy number variations (CNVs) at a single-cell level. We observed significant inter-tumor heterogeneity and started to deconstruct the genomic architecture of individual patients. Using experimentally validated gene sets, we determined the cell cycle state of individual cells and identified transcriptional programs related to the cell cycle as significant bias in publically available bulk RNA-sequencing data. Principal component analysis revealed the expression of a stem-ness signature, including CD133, ALDH1A and AXL, in a sub-set of non-cycling cells. An important driver of transcriptional heterogeneity common to patients included in this study was the expression of gene sets related to inflammatory pathways, such as the NFkB and JAK/STAT pathways. Hierarchical clustering of 42 spheroid profiles identified four major clusters, including a highly “inflamed” phenotype. Therapeutic inhibition of the STAT pathway abrogated the capacity of spheroid formation on an ultra-low attachment surface, indicating its importance for metastasis. We have successfully isolated thousands of individual cells for single-cell profiling from PDX models treated with carboplatin. These cells were collected at three time points, including at the MRD stage. We have successfully sequenced 100 cells from this collection and were able to generate whole-transcriptome data comparable to that of freshly isolated patient cells and thousands of single cells are currently undergoing sequencing. CONCLUSION: We have successfully applied single-cell RNA-sequencing to patient-derived ovarian cancer cells and PDX-models. Single-cell transcriptomes enabled inference of genomic information, genetic and transcriptional heterogeneity, cell cycle state, and programs related to stem-ness and inflammation, providing a unique and comprehensive perspective on ovarian cancer cell states. Ongoing profiling of carboplatin-resistant cells captured at the minimal residual disease stage in PDX-models will provide a unique opportunity to understand treatment resistance which ultimately leads to cancer recurrence. Citation Format: Benjamin Izar, Elizabeth Stover, Itay Tirosh, Asaf Rotem, Parin Shah, Chris Rodman, Sanjay Prakadan, Marc Wadsworth, Mei-Ju Su, Rachel Leeson, Sangeetha Palakurthi, Joyce Liu, Ursula Matulonis, Alex Shalek, Orit Rozenblatt-Rosen, Aviv Regev, Levi Garraway. SINGLE–CELL RNA–SEQUENCING OF PATIENT–DERIVED OVARIAN CANCER CELLS AND PATIENT–DERIVED XENOGRAFT MODELS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP19.

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells.

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Whereas the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reversed Phase Protein Array and mass cytometry ‘CyTOF’ analyses demonstrated that ARV-825 caused greater perturbations in mRNA and protein expressions than OTX015 in sAML cells. Specifically, compared to OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, pSTAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared to OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against the established and PD-CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Superior Lethal Activity of Novel BET Protein Proteolysis Targeting Chimera (BETP-PROTACs) Versus Betp Bromodomain Inhibitor (BETi) Against Post-Myeloproliferative Neoplasm (MPN) Secondary (s) AML Cells

Hematopoietic stem/progenitor cells (HPCs) of BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 & 2 inhibitor (JAKi), which is currently used in the therapy of MPN-MF. Treatment with R confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Prolonged exposure to R may also lead to a loss of response, causing the emergence of JAKi-resistant (JIR) sAML cells. Although they lack additional JAK2 mutations, JIR cells exhibit reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TK). Sequential genomic assessments in pre- and post-sAML transformation have revealed mutations in TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, SETBP1 and TP53 genes. Here, we determined that treatment with BETi, e.g., JQ1 or OTX015, which disrupts the binding of BRD4 with acetylated chromatin, significantly inhibits growth and induces apoptosis of JAKi-sensitive cultured, including those that express JAK2 V617F and mutant TP53, e.g., HEL92.1.7 and SET2, and patient derived (PD) CD34+ sAML cells. Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. Co-treatment with BETi and R synergistically induced apoptosis of cultured and PD CD34+ sAML cells. As compared to treatment with vehicle control, JQ1 or R treatment alone, co-treatment with JQ1 and R significantly improved the median survival of the immune-depleted mice engrafted with HEL92.1.7 cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the activity of c-MYC, NFkB and several other transcription factors. Therefore, we compared the anti-sAML activity of the novel BETP-PROTACs (BET protein-proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.), which degrade BETPs (including BRD4), with the BETi OTX015, against cultured and PD CD34+ sAML cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BETPs. At equimolar concentrations (100 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary sAML cells (p < 0.05), while sparing the CD34+ normal hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased BRD4 levels, BETP-PROTACs caused efficient and prolonged depletion of the levels of BETPs, including BRD4 (> 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. Western analyses showed that BETP-PROTAC versus BETI treatment also caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry approach showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-RB, while inducing p21 levels in the phenotypically characterized CD34+ sAML stem/progenitor cells, based on their high expression of CD90, CD244, CD123+ and TIM3Fc+. Compared to treatment with each agent alone, co-treatment with ARV-825 and R (100 to 1000 nM) was synergistically more lethal against the cultured and PD CD34+ sAML cells. We have isolated JAKi-resistant HEL92.1.7 cells (> 10-fold resistant to R; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAKi. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to BETP-PROTAC. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 is synergistically lethal against JAKi sensitive and JIR sAML cells. These findings strongly support the in vivo testing of the BETP-PROTACs alone and in combinations against post-MPN sAML. These studies are underway in our laboratory and will be presented at the ASH meetings. DisclosuresRaina:Arvinas, LLC: Employment. Coleman:Arvinas, LLC: Employment. Winkler:Arvinas, LLC: Employment. Qian:Arvinas, LLC: Employment. Crew:Arvinas, LLC: Employment. Shen:Arvinas, LLC: Employment.

Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) Exert Potent Single Agent and Synergistic Activity with Ibrutinib and Venetoclax Against Human Mantle Cell Lymphoma Cells

Mantle Cell Lymphoma (MCL) exhibits pathogenetic mutations or deletion of RB1, ATM, p53, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2. Activated B cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific ’transcriptome’ that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton’s tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary refractory/resistant disease with a one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, despite ibrutinib treatment, sustain classical or alternative NFkB signaling and transcriptional activity, as well as confer resistance to ibrutinib in MCL. We previously reported that the BET protein (BETP) bromodomain inhibitors (BETis), which disrupt the binding of BRD4 with acetylated chromatin, inhibit the in vitro growth and induce apoptosis of cultured and patient-derived (PD) primary MCL cells. This was associated with BETi-mediated attenuation of c-MYC, BCL2, CDK4/6 as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, Bcl-xL, IL10, TNFα and BTK. Concomitantly, BETi treatment induced HEXIM1, p21, p27 and NOXA levels in MCL cells. Co-treatment with BETi and ibrutinib was synergistically lethal and improved the median survival of the immune-depleted mice engrafted with human MCL cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the deregulated transcriptional activity of c-MYC, NFkB and other transcription factors. Here, we compared the anti-MCL activity of the novel BETP-PROTACs (proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) that degrade BRD4 with the BETi OTX015 against cultured and primary MCL cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BET proteins including BRD4. At equimolar concentrations (10 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary MCL cells (p < 0.01), while sparing the CD19+ normal B and CD34+ hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased, BETP-PROTACs markedly attenuated (> 90%) the levels of BRD4 in the MCL cells. BETP-PROTAC treatment caused more profound up and down regulation of mRNA and protein expressions, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. BETP-PROTAC treatment also caused greater and more sustained depletion of c-MYC, CDK4/6, PIM1, cyclin D1, as well as of the NFkB transcriptional targets Bcl-xL, XIAP, MCL1 and BTK, while concomitantly inducing the level of NOXA and p21. As compared to treatment with OTX015, ARV-771 treatment dramatically inhibited the growth and improved survival of NSG mice engrafted with luciferase-transduced, ibrutinib-resistant, Z138 MCL cells. Also, notably, co-treatment of ARV-825 or ARV-771 with ibrutinib or the BCL2-antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of cultured and primary MCL cells. We have also generated, ex vivo, ibrutinib-resistant (e.g., Mino/IR and JeKo1/IR) and ibrutinib-persister/resistant (Mino/IPR) cultured MCL cells, with >10-fold higher IC50 value for ibrutinib than the parental MCL cells. BETP-PROTAC treatment more potently induced lethality than BETi in the Mino/IR and Mino/IPR cells, associated with attenuation of c-MYC, BCL2, CDK4/6 and NFkB target gene expressions including BTK. BETP-PROTAC and BETi also induced synergistic lethality with venetoclax and palbociclib against the Mino/IR, Mino/IPR and Z138 MCL cells. These findings underscore the superior in vitro and in vivo activity of BETP-PROTACs versus BETi against ibrutinib-sensitive and ibrutinib-refractory MCL cells, as well as highlight a promising new class of agents to be developed for the therapy of human MCL. DisclosuresRaina:Arvinas, LLC: Employment. Coleman:Arvinas, LLC: Employment. Winkler:Arvinas, LLC: Employment. Qian:Arvinas, LLC: Employment. Crew:Arvinas, LLC: Employment. Shen:Arvinas, LLC: Employment. Wang:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; BeiGene: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Onyx: Research Funding; Celgene: Research Funding.