Identification of Novel Synthetic Lethal Partners of NAMPT Inhibitor By CRISPR-Cas9 Screens in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematopoietic neoplasm arising from the clonal expansion of myeloid progenitors resulting in bone marrow failure. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate‐limiting enzyme which regulates the generation of the metabolic enzyme substrate NAD+. NAD+ is key in several survival pathways in AML such as DNA repair and metabolic pathways. AML subtypes differ vastly in their inherent DNA repair and metabolic capacities. Since NAMPT is essential in single and double strand DNA repair, NAMPT inhibitors (NAMPTis) were used to block DNA repair as single agents or in combinational therapies in solid tumors. However the clinical experience with the first generation of NAMPTis suggests that higher doses will be needed in order to achieve efficacy, which are associated with high toxicity. We hypothesize that identification of critical synthetic lethal partners for NAMPTi will increase efficacy with lower doses. To this end, we conducted genome-wide CRISPR KO (loss-of-function) screens by using two libraries, GeCKO and Brunello, and identified, along with genes reported essential for AML survival, 16 genes whose knockout displays synergism or resistance, after a NAMPTi (KPT-9274) treatment in AML cells. According to their gene ontology (GO) functions, these co-essential candidates are involved in DNA damage repair and metabolism. Genetic depletion of co-essential genes, SIRT6, HDAC8 and DCPS, sensitizes AML cell lines, MOLM13, Kasumi-1 and MV4-11, to KPT-9274 treatment. In addition, preclinical inhibitor for HDAC8 (PCI-34051), in combination with KPT-9274, synergistically decreased AML primary cell survival in a dose-dependent manner and decreased colony formation in AML patient samples across multiple genotypes with a minimal decrease in colony formation of normal CD34+ hematopoietic cells. In addition, drug combo treatment of AML cells decreased re-plating capacity, implying attenuated self-renewal capacity. Drug combo also increased apoptotic cells as measured by Annexin/PI staining and induced myeloid differentiation as detected by CD45+CD34- staining. In DNA homologous recombination (HR) pathway, SIRT6 and HDAC8 deacetylate CtIP and Rad51 to promote DNA end resection at double strand breaks. Since NAMPT inhibition blocks the generation of NAD+ thus compromising downstream PARP1 mediated base excision repair (BER) as well as the Sirtuin1 (Sirt1) mediated, KU- dependent NHEJ and atypical Rad51-independent HR repair, we expect that inhibiting these HR pathway components (like SIRT6 and HDAC8) may shut down these compensatory repair pathways and sensitize AML subtypes to NAMPT inhibition. Indeed, Western blotting shows drug combo increased acetyl-P53, acetyl-Rad51, phosphor-γH2Ax and Chk1 compared NAMPT inhibition alone. The in vivo efficacy of these combinatorial therapies has been investigated on PDX mouse models established in our laboratory. We demonstrated that the dug combo successfully eliminated engrafted AML cells and conferred survival advantages to mice. Our study provides evidence that co-essential genes, SIRT6, HDAC8 and DCPS, are synthetic lethal partners for NAMPT inhibition to target drug-resistant AML cells, with minimal toxicity towards normal cells. Disclosures Byrd: BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Acerta: Research Funding; Janssen: Consultancy, 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.

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Genomic Analysis of Cellular Hierarchy in Acute Myeloid Leukemia Using Ultrasensitive LC-FACS-Seq

Blood ◽

10.1182/blood-2019-125285 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 186-186

Author(s):

Caner Saygin ◽

Arletta Lozanski ◽

Tzyy-Jye Doong ◽

Shelley Orwick ◽

Deedra Nicolet ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Research Funding ◽

Ohio State University ◽

Median Frequency ◽

State University ◽

University Patents ◽

Pre Treatment ◽

Acute Myeloid

Normal hematopoiesis is organized in a hierarchical manner and it has been hypothesized that acute myeloid leukemia (AML) is organized in a similar way with leukemia-initiating cells (LIC) at the top of the hierarchy, giving rise to more differentiated blasts to sustain AML. Therefore, elimination of LIC population is critical for cure. This may be accomplished via novel molecular targeted therapies. The mutational composition of LIC and non-LIC compartments in AML has not been fully elucidated and could provide new insights into biology and treatment. We investigated the distribution and variant allelic frequencies (VAFs) of recurrent gene mutations within these compartments in newly diagnosed CD34+ AML patients (pts). We studied a total of 88 pts. CD34- AML cases, defined as <5% positivity on blasts, were excluded. Pre-treatment bone marrow or apheresis samples were sorted and sequenced with our ultrasensitive limited cell (LC)-FACS-seq method. First, we gated on CD45dimLin- leukemic population, followed by isolation of 300 cells from CD34+CD38- (LIC), CD34+CD38+ (non-LIC) and CD34- compartments. To compare with the bulk population, DNA was extracted from 500,000 CD45dimLin- leukemic blasts. All samples were sequenced with a 27-gene targeted panel. Extreme Limited Dilution Analysis (ELDA) platform was used for colony formation assays and estimation of stem cell frequencies. Clinical characteristics are summarized in Table 1. The median frequency of the LIC population was 0.5% (range, 0.01% - 69%). The prevalence of high LIC frequency (≥0.5%) was significantly higher in pts with adverse risk (AR) AML, as compared to intermediate (IR) and favorable risk groups (94% vs 34% vs 16%, respectively, p<.001). When compared to pts with low LIC frequency (<0.5%), those with high LIC frequency had worse overall survival (median, 9 months vs not reached, p=.003) and relapse-free survival (median, 4 vs 15 months, p=.01). In 10 pts who had serial relapse samples, LIC frequencies were increased at the time of relapse (p=.03). We re-validated the commonly used LIC markers with ELDA of primary AML cells. In one IR sample, stem cell frequencies in sorted CD34+CD38-, CD34+CD38+ and CD34- compartments were 1:3, 1:15 and 1:16, respectively (p<.001). In one AR sample, stem cell frequencies were 1:1, 1:8, and 1:12, respectively (p<.001). Using these markers, LICs and non-LICs were enriched and sequenced. The average number of mutations detected by sequencing of bulk samples was significantly lower than sorted LIC (3.17 vs 3.75, p<.05) and non-LIC (3.17 vs 3.96, p<.001) populations indicating the higher sensitivity of our method in detecting subclonal mutations. Mean VAFs were similar between LIC and non-LIC populations for NPM1 (42% vs 47%), DNMT3A (37% vs 41%), IDH1 (41% vs 48%), IDH2 (43% vs 48%), and U2AF1 (37% vs 42%) mutations. Mutations involving signaling pathways were more frequent in non-LICs, including FLT3-TKD (12% vs 23%, p<.01), NRAS (17% vs 26%, p<.01) and KRAS (13% vs 19%, p<.05) mutations, which might be explained by their later acquisition during AML development. In addition, among 22 pts with CEBPA mutation, 16 (73%) harbored the mutation exclusively in non-LICs. Finally, 13 pts with TP53 mutations had different VAFs between compartments. Among 4 pts who had doubling of VAF from LIC to non-LIC compartment, 3 had subclones with del(17p) in LIC pool detected by FISH. LIC subclones harboring both del(17p) and TP53 mutation (i.e. loss of heterozygosity) propagated to drive leukemia. Relapse samples obtained from 6 pts were analyzed and compared with diagnosis. In all cases, we could identify LIC clones that persisted after chemotherapy and led to relapse (see example in Figure). Similarly, 3 pts who were primary refractory showed persistence of LIC clones that were resistant to treatment. On the contrary, 6 pts in whom LIC clones could be eradicated with treatment did not experience disease recurrence. LICs exist at a very low frequency in pre-treatment AML samples. The mutational composition of LIC-enriched compartment shows differences from blasts constituting the bulk of leukemia, which is consistent with the sequence of mutations observed during the evolution of AML. LC-FACS-seq is an ultrasensitive method to detect mutations in a tiny population of residual LICs in pts at remission. Therapies targeting mutations that are concentrated in LICs may re-shape the clonal hierarchy and impact on disease course. Disclosures Behbehani: Fluidigm corporation: Other: Travel funding. Byrd:Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding.

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SIRT6 Inhibition As a Novel Approach for Treating Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.5222.5222 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5222-5222

Author(s):

Michele Cea ◽

Antonia Cagnetta ◽

Debora Soncini ◽

Paola Minetto ◽

Davide Lovera ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Dna Damage ◽

Dna Repair ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Allogeneic Bone ◽

A Genome ◽

Acute Myeloid

Abstract Currently available therapeutics against Acute Myeloid Leukemia (AML) have improved patient outcome. However, resistance develops even to novel therapies and patient overall survival remains low, especially for patients who are not eligible for allogeneic bone marrow transplantation. Therefore, there is an urgent need to overcome the biologic mechanisms underlying drug resistance in AML, to enhance the efficacy of existing treatments and to facilitate the design of novel approaches. Recently, our group has demonstrated that SIRT6, a NAD+-dependent histone deacetylase involved in genome maintenance, is frequently up-regulated in Multiple Myeloma and its targeting induces cancer cell killing (Cea M. et al, Blood 2016). Furthermore, gene expression analyses performed by our groups show that SIRT6 is also up-regulated in AML and confers poor prognosis in a series of 200 primary AML cases from our Hematology Clinic. Thus, these data suggested a role for SIRT6 in AML biology. High SIRT6 expression was typically observed in AML cell lines characterized by constitutive DNA damage and intense replicative stress. Likewise, primary AML cases exhibiting an intermediate-to-high chromosome instability (CIN) gene expression signature were also those with the highest SIRT6 expression, and worst prognosis. Subsequent studies demonstrated that SIRT6 silencing or its chemical inhibition, as observed in Multiple Myeloma exacerbates DNA damage in response to genotoxic agents, sensitizing AML cells to cytarabine (ARA-C) and idarubicin in vitro. Overall, enhancing genotoxic stress while concomitantly blocking DNA double-strand breaks (DSBs) repair response, may represent an innovative strategy to increase chemosensitivity of AML cells. Mechanistic studies revealed that SIRT6 acts as a genome guardian in AML cells by binding DNA damage sites and activating DNA-PKcs and CtIP by deacetylation, which in turn promotes DNA repair. Overall our data suggest that genomic instability is present in all hematologic malignancies including AML. Strategies aimed to shift the balance towards high DNA damage and reduced DNA repair by SIRT6 inhibition can decrease AML growth and may benefit patients with otherwise unfavorable outcomes. Disclosures Gobbi: Gilead: Honoraria; Takeda: Consultancy; Janssen: Consultancy, Honoraria; Roche: Honoraria; Celgene: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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Discovery of a Novel Dihydroorotate Dehydrogenase Inhibitor That Induces Differentiation and Overcomes Ara-C Resistance of Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood-2019-121869 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2663-2663

Author(s):

Satoshi Kitazawa ◽

Yukiko Ishii ◽

Keiko Makita-Suzuki ◽

Koichi Saito ◽

Kensuke Takayanagi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Oral Administration ◽

Myeloid Leukemia ◽

Research Funding ◽

Dihydroorotate Dehydrogenase ◽

Once Daily ◽

Hl60 Cells ◽

Pyrimidine Nucleotide ◽

Acute Myeloid

Cancer initiating cells (CIC) are suggested to be responsible for drug resistance and cancer relapse that are associated with poor prognosis. Therefore, drugs effective for CIC could fulfill an unmet clinical need. We performed a drug screen with chemical libraries to find out new compounds which specifically eradicated CIC established in the previous report (Yamashita et al., Cancer Research, 2015). We obtained compounds with a carboxylic acid skeleton as hit compounds. Interestingly, FF1215T, one of the hit compounds, was shown to inhibit growths of CIC by decreasing intracellular pyrimidine nucleotide levels. Finally, we identified dihydroorotate dehydrogenase (DHODH), which was essential for de novo pyrimidine synthesis as the target of the hit compounds in a ligand fishing assay. FF1215T inhibited DHODH enzymatic activity with the 50% inhibitory concentration value of 9 nM, which showed greater potency than well-known DHODH inhibitors brequinar (12 nM), teriflunomide (262 nM), and vidofludimus (141 nM). Growing evidence suggests that DHODH is considered to be a promising target to overcome a differentiation blockade of acute myeloid leukemia (AML) cells (Sykes et al., Cell, 2016).Therefore, we explored the effect of FF1215T on AML growth and differentiation. FF1215T demonstrated growth inhibitory effect in multiple human AML cell lines such as U937, MOLM13, HL60, and MV4-11 with the 50% growth inhibition values of 90-170 nM. FF1215T decreased intracellular pyrimidine nucleotide levels, induced DNA damage marker γ-H2AX possibly due to the replication stress, and finally led to apoptosis in HL60 cells. Cell cycle analysis revealed that FF1215T treatment arrested HL60 and THP1 cells at S phase and increased sub-G1 population in these cells. In addition, our DHODH inhibitors induced upregulation of cell-surface CD11b and CD86, which are monocyte and macrophage differentiation markers, morphological changes, and phagocytic activities in several AML cells, indicating differentiation of AML cells toward monocyte and macrophage by DHODH inhibition. FF1215T also depleted UDP-GlcNAc, a substrate for Protein O-GlcNAcylation, and diminished global O-GlcNAcylation and O-GlcNAcylated protein expressions such as c-Myc, SOX2, and OCT4, which play important roles in maintenance and self renewal of stem cells. We also found that our DHODH inhibitors induced CD11b and CD86, and increased the ratio of macrophage-like cells in primary patient-derived AML cells and these effects were rescued by uridine supplementation (Fig). Inhibitions of colony formations of primary AML cells were also shown after 14 days of FF1215T treatment. In exploring the value of DHODH inhibitors in the clinic, we identified that our DHODH inhibitors worked to overcome the resistance of standard therapy Ara-C. Our DHODH inhibitors were effective against Ara-C-resistant models of HL60 cells as well as HL60 parental cells. Notably, our DHODH inhibitors synergistically inhibited growths of Ara-C-resistant THP1 cells and enhanced CD11b upregulation of THP1 cells when combined with Ara-C by activating conversion of Ara-C to its active form Ara-CTP. Next, we optimized the hit compounds and identified an orally available DHODH inhibitor FF14984T that achieved high and prolonged plasma concentrations in vivo. Oral administration of 10 and 30 mg/kg FF14984T once daily for 10 days exhibited significant anti-tumor effects in mice xenografted with HL60 cells. These treatments showed strong reduction of CTP in tumor and induction of DHO in tumor and plasma. When 30 mg/kg FF14984T was orally administrated to orthotropic MOLM13-xenografted mice once daily for 12 days, hCD45+ cells proportions in bone marrow were decreased whereas hCD11bhigh/hCD45+ ratio increased, indicating that FF14984T induced AML differentiation in vivo. Finally, oral administration of 30 mg/kg FF14984T once daily significantly prolonged survival of mice in U937 orthotropic models. Taken together, we developed a novel potent DHODH inhibitor FF14984T that induced cellular differentiation and anti-leukemic effects on cell lines and primary AML cells. FF14984T is possibly a promising therapeutic option for Ara-C-resistant AML patients that can also benefit from the combination therapy of FF14984T and Ara-C. Identifying the precise mechanism of AML differentiation by DHODH inhibitor and its effects on CIC are currently ongoing. Disclosures Kitazawa: FUJIFILM Corporation: Employment. Ishii:FUJIFILM Corporation: Employment. Makita-Suzuki:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Takayanagi:FUJIFILM Corporation: Employment. Sugihara:FUJIFILM Corporation: Employment. Matsuda:FUJIFILM Corporation: Employment. Yamakawa:FUJIFILM Corporation: Employment. Tsutsui:FUJIFILM Corporation: Employment. Tanaka:FUJIFILM Corporation: Employment. Hatta:FUJIFILM Corporation: Research Funding. Natsume:FUJIFILM Corporation: Research Funding. Kondo:FUJIFILM Corporation: Research Funding. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Perseus Proteomics Inc.: Research Funding.

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PKR-like Endoplasmic Reticulum Kinase Mediates Apoptosis Induced By Pharmacological AMP-Activated Protein Kinase Activation in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-123364 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2552-2552

Author(s):

Laury Poulain ◽

Adrien Grenier ◽

Johanna Mondesir ◽

Arnaud Jacquel ◽

Claudie Bosc ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Endoplasmic Reticulum Stress Response ◽

Ampk Activation ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Amp Activated Protein Kinase ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a myeloid progenitor-derived neoplasm of poor prognosis, particularly among the elderly, in whom age and comorbidities preclude the use of intensive therapies. Novel therapeutic approaches for AML are therefore critically needed. Adenosine monophosphate (AMP) activated protein kinase (AMPK) is a pleiotropic serine/threonine kinase promoting catabolism that represses anabolism and enhances autophagy in response to stress1. AMPK heterotrimers comprise catalytic α- and regulatory β- and γ-subunits, the latter harboring binding sites for AMP. Targets of AMPK include a host of metabolic pathway enzymes mediating carbohydrate, lipid and protein synthesis and metabolism. Accumulating evidence implicates AMPK in cancer biology, primarily as a tumor suppressor, although minimal AMPK activity may also be required for cancer cell growth under stress conditions2,3. Pharmacological activation of AMPK thus represents an attractive new strategy for targeting AML. We previously used the selective small molecule AMPK activator GSK621 to show that AMPK activation induces cytotoxicity in AML but not in normal hematopoietic cells, contingent on concomitant activation of the mammalian target of rapamycin complex 1 (mTORC1)4. However, the precise mechanisms of AMPK-induced AML cytotoxicity have remained unclear. We integrated gene expression profiling and bioinformatics proteomic analysis to identify the serine/threonine kinase PERK - one of the key effectors of the endoplasmic reticulum stress response - as a potential novel target of AMPK. We showed that PERK was directly phosphorylated by AMPK on at least two conserved residues (serine 439 and threonine 680) and that AMPK activators elicited a PERK/eIF2A signaling cascade independent of the endoplasmic reticulum stress response in AML cells. CRISPR/Cas9 depletion and complementation assays illuminated a critical role for PERK in apoptotic cell death induced by pharmacological AMPK activation. Indeed, GSK621 induced mitochondrial membrane depolarization and apoptosis in AML cells, an effect that was mitigated when cells were depleted of PERK or expressed PERK with a loss of function AMPK phosphorylation site mutation. We identified the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) as a downstream target of the AMPK/PERK pathway, as its expression was enhanced in PERK knockdown AML cells. Moreover, selective pharmacologic activation of ALDH2 by the small molecule ALDA-1 recapitulated the protective effects of PERK depletion in the face of pharmacological AMPK activation. Corroborating the impact of the AMPK/PERK axis on mitochondrial apoptotic function, BH3 profiling showed marked Bcl-2 dependency in AML cells treated with GSK621. This dependency was abrogated in PERK-depleted cells, suggesting a role for PERK in mitochondrial priming to cell death. In vitro drug combination studies further demonstrated synergy between the clinical grade Bcl-2 inhibitor venetoclax (ABT-199) and each of four AMPK activators (GSK621, MK-8722, PF-06409577 and compound 991) in multiple AML cell lines. Finally, the addition of GSK621 to venetoclax enhanced anti-leukemic activity in primary AML patient samples ex vivo and in humanized mouse models in vivo. These findings together clarify the mechanisms of cytotoxicity induced by AMPK activation and suggest that combining pharmacologic AMPK activators with venetoclax may hold therapeutic promise in AML. References 1. Lin S-C, Hardie DG. AMPK: Sensing Glucose as well as Cellular Energy Status. Cell Metabolism. 2018;27(2):299-313. 2. Hardie DG. Molecular Pathways: Is AMPK a Friend or a Foe in Cancer? Clinical Cancer Research. 2015;21(17):3836-3840. 3. Jeon S-M, Hay N. The double-edged sword of AMPK signaling in cancer and its therapeutic implications. Arch. Pharm. Res. 2015;38(3):346-357. 4. Sujobert P, Poulain L, Paubelle E, et al. Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia. Cell Rep. 2015;11(9):1446-1457. Figure Disclosures Tamburini: Novartis pharmaceutical: Research Funding; Incyte: Research Funding.

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Myelodysplastic Syndrome with Excess Blasts and Secondary Acute Myeloid Leukemia: Same Disease with Different Blast Count

Blood ◽

10.1182/blood-2019-124977 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2692-2692

Author(s):

Xueyan Chen ◽

Megan Othus ◽

Brent L Wood ◽

Roland B. Walter ◽

Pamela S. Becker ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Mutational Analysis ◽

World Health ◽

Secondary Aml ◽

Data Safety ◽

Monitoring Committee ◽

Mutational Hotspots ◽

Acute Myeloid

Introduction: The World Health Organization (WHO) diagnoses acute myeloid leukemia (AML) if ≥20% myeloid blasts are present in peripheral blood or bone marrow. Consequently a patient with even 19% blasts is often ineligible for an "AML study". A less arbitrary means to define "AML" and myelodysplastic syndromes ("MDS") emphasizes biologic features. Here, focusing on patients with WHO-defined MDS with excess (5-19%) blasts (MDS-EB) or AML with myelodysplasia-related changes (AML-MRC) or therapy-related (t-AML) (WHO defined secondary AML), we compared morphologic blast percentage (MBP) with the frequency of mutations in genes belonging to different functional groups, and with the variant allele frequency (VAF) for individually mutated genes. Methods: 328 adults with WHO-defined AML (de novo and secondary; n=149) or MDS (n=179) and with mutational analysis by next-generation sequencing (NGS) performed at the University of Washington Hematopathology Laboratory between 2015-2017 were included. Of these, 86 had MDS-EB and 49 had secondary AML. Mutational analysis was performed using a customized, amplicon-based assay, TruSeq Custom Amplicon (Illumina, San Diego, CA). Custom oligonucleotide probes targeted specific mutational hotspots in ASXL1, CBL, CEBPA, CSF3R, EZH2, FBXW7, FGFR1, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KMT2A, KRAS, MAP2K1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PTEN, RB1, RUNX1, SF3B1, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, WT1, and ZRSR2. VAF ≥5% was required to identify point mutations. Spearman's correlation coefficient was used to examine the relation between VAF of individually mutated genes and MBP. The Mann Whitney test served to compare the distribution of VAF in AML (≥20% blasts) vs. MDS (<20% blasts), before and after exclusion of subgroups as described below. Fisher's exact test was used to compare incidence of mutations. Results: 96% of cases had ≥one mutation in the 36 genes tested using NGS. Considering all 328 patients, mutations in tumor suppressor and cohesin complex genes were similarly frequent in MDS and AML, whereas spliceosomal genes, in particular SF3B1 and SRSF2, were more frequently mutated in MDS than in AML (46% vs. 26%, p<0.001). Mutations in epigenetic modifiers were more common in AML than MDS (54% vs. 42%, p= 0.035) as were transcription factor mutations (52% vs. 28%, p<0.001). However comparisons limited to MDS-EB vs. AML-MRC/t-AML, indicated the differences observed when comparing all MDS and all AML were less apparent, both statistically and more perhaps importantly with respect to observed frequencies. For example, spliceosomal gene mutations were found in 35% in MDS-EB and 27% in AML-MRC/t-AML (p=0.34) vs. 46% and 26% in all MDS and all AML. NPM1 mutations were detected in only 8% of AML-MRC/t-AML vs. 3% in MDS-EB but 29% for all AML. Results were analogous with FLT3 ITD, FLT3 TKD, and JAK2 mutations. Examining 20 individually mutated genes detected in ≥ 10 patients only with SRSF2 (p=0.04), did distribution of VAF differ statistically according to whether blast percentage was <20% versus ≥20%. Conclusions: The similar prevalence of mutations in different functional categories in MDS-EB and AML-MRC/t-AML suggests these entities are two manifestations of the same disease. We believe it appropriate to combine these WHO entities allowing patients in each to be eligible for both AML and MDS trials. Disclosures Othus: Glycomimetics: Other: Data Safety and Monitoring Committee; Celgene: Other: Data Safety and Monitoring Committee. Walter:Amgen: Consultancy; Boston Biomedical: Consultancy; Agios: Consultancy; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy; Seattle Genetics: Research Funding; Amphivena Therapeutics: Consultancy, Equity Ownership; Boehringer Ingelheim: Consultancy; Aptevo Therapeutics: Consultancy, Research Funding. Becker:Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; The France Foundation: Honoraria.

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Prospective Identification of Acute Myeloid Leukemia Patients Who Benefit from Gene-Expression Based Risk Stratification

Blood ◽

10.1182/blood-2019-129519 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1397-1397

Author(s):

Diego Chacon ◽

Ali Braytee ◽

Yizhou Huang ◽

Julie Thoms ◽

Shruthi Subramanian ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Risk Stratification ◽

Genetic Risk ◽

Myeloid Leukemia ◽

Research Funding ◽

Risk Groups ◽

High Fidelity ◽

Improve Outcome ◽

Acute Myeloid

Background: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy and risk stratification based on genetic and clinical variables is standard practice. However, current models incorporating these factors accurately predict clinical outcomes for only 64-80% of patients and fail to provide clear treatment guidelines for patients with intermediate genetic risk. A plethora of prognostic gene expression signatures (PGES) have been proposed to improve outcome predictions but none of these have entered routine clinical practice and their role remains uncertain. Methods: To clarify clinical utility, we performed a systematic evaluation of eight highly-cited PGES i.e. Marcucci-7, Ng-17, Li-24, Herold-29, Eppert-LSCR-48, Metzeler-86, Eppert-HSCR-105, and Bullinger-133. We investigated their constituent genes, methodological frameworks and prognostic performance in four cohorts of non-FAB M3 AML patients (n= 1175). All patients received intensive anthracycline and cytarabine based chemotherapy and were part of studies conducted in the United States of America (TCGA), the Netherlands (HOVON) and Germany (AMLCG). Results: There was a minimal overlap of individual genes and component pathways between different PGES and their performance was inconsistent when applied across different patient cohorts. Concerningly, different PGES often assigned the same patient into opposing adverse- or favorable- risk groups (Figure 1A: Rand index analysis; RI=1 if all patients were assigned to equal risk groups and RI =0 if all patients were assigned to different risk groups). Differences in the underlying methodological framework of different PGES and the molecular heterogeneity between AMLs contributed to these low-fidelity risk assignments. However, all PGES consistently assigned a significant subset of patients into the same adverse- or favorable-risk groups (40%-70%; Figure 1B: Principal component analysis of the gene components from the eight tested PGES). These patients shared intrinsic and measurable transcriptome characteristics (Figure 1C: Hierarchical cluster analysis of the differentially expressed genes) and could be prospectively identified using a high-fidelity prediction algorithm (FPA). In the training set (i.e. from the HOVON), the FPA achieved an accuracy of ~80% (10-fold cross-validation) and an AUC of 0.79 (receiver-operating characteristics). High-fidelity patients were dichotomized into adverse- or favorable- risk groups with significant differences in overall survival (OS) by all eight PGES (Figure 1D) and low-fidelity patients by two of the eight PGES (Figure 1E). In the three independent test sets (i.e. form the TCGA and AMLCG), patients with predicted high-fidelity were consistently dichotomized into the same adverse- or favorable- risk groups with significant differences in OS by all eight PGES. However, in-line with our previous analysis, patients with predicted low-fidelity were dichotomized into opposing adverse- or favorable- risk groups by the eight tested PGES. Conclusion: With appropriate patient selection, existing PGES improve outcome predictions and could guide treatment recommendations for patients without accurate genetic risk predictions (~18-25%) and for those with intermediate genetic risk (~32-35%). Figure 1 Disclosures Hiddemann: Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Metzeler:Celgene: Honoraria, Research Funding; Otsuka: Honoraria; Daiichi Sankyo: Honoraria. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Beck:Gilead: Research Funding.

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Mini- Vs. Regular-Dose CLAG-M (Cladribine, Cytarabine, G-CSF, and Mitoxantrone) in Medically Less Fit Adults with Newly-Diagnosed Acute Myeloid Leukemia (AML) and Other High-Grade Myeloid Neoplasms

Blood ◽

10.1182/blood-2019-123542 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1364-1364 ◽

Cited By ~ 1

Author(s):

Anna B. Halpern ◽

Megan Othus ◽

Kelda Gardner ◽

Genevieve Alcorn ◽

Mary-Elizabeth M. Percival ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Intensive Therapy ◽

Treatment Intensity ◽

High Grade ◽

Intensive Chemotherapy ◽

Myeloid Neoplasms ◽

Acute Myeloid

Background: Optimal treatment for medically less fit adults with acute myeloid leukemia (AML) remains uncertain. Retrospective data suggest intensive therapy may lead to better outcomes in these patients. However, these findings must be interpreted cautiously because of the possibility of selection bias and other confounders. Ideally, the optimal treatment intensity is defined via randomized trial but whether patients and their physicians are amenable to such a study is unknown. We therefore designed a trial (NCT03012672) to 1) evaluate the feasibility of randomization between intensive and non-intensive therapy in this population and 2) examine the impact of treatment intensity on response rate and survival. We used CLAG-M as high-dose cytarabine-based intensive induction therapy. Rather than selecting different classes of drugs in the 2 treatment arms- which may have different modes of action and therefore confound the question of treatment intensity - we used reduced-dose ("mini") CLAG-M as the non-intensive comparator. Methods: Adults ≥18 years were eligible if they had untreated AML or high-grade myeloid neoplasms (≥10% blasts in blood or marrow) and were medically less fit as defined by having a "treatment related mortality" (TRM) score of ≥13.1, corresponding to a >10-15% 28-day mortality with intensive chemotherapy. Left ventricular ejection fraction ≤45% was the only organ function exclusion. Patient-physician pairs were first asked if they were amenable to randomized treatment allocation. If so, they were randomized 1:1 to mini- vs. regular-dose CLAG-M. If not, in order to evaluate our secondary endpoints, the patient or physician could choose the treatment arm and still enroll on study. Patients and physicians then completed surveys elucidating their decision-making processes. Up to 2 induction courses were given with mini- vs. regular-dose CLAG-M: cladribine 2 or 5 mg/m2/day (days 1-5), cytarabine 100 or 2,000 mg/m2/day (days 1-5), G-CSF 300 or 480µcg/day for weight </≥76kg in both arms (days 0-5), and mitoxantrone 6 or 18 mg/m2/day (days 1-3). CLAG at identical doses was used for post-remission therapy for up to 4 (regular-dose CLAG) or 12 (mini-CLAG) cycles. The primary endpoint was feasibility of randomization, defined as ≥26/50 of patient-physician pairs agreeing to randomization. Secondary outcomes included rate of complete remission (CR) negative for measurable ("minimal") residual disease (MRD), rate of CR plus CR with incomplete hematologic recovery (CR+CRi), and overall survival (OS). Results: This trial enrolled 33 patients. Only 3 (9%) patient/physician pairs agreed to randomization and thus randomization was deemed infeasible (primary endpoint). Eighteen pairs chose mini-CLAG-M and 12 regular-dose CLAG-M for a total of 19 subjects in the lower dose and 14 subjects in the higher dose arms. The decision favoring lower dose treatment was made largely by the physician in 5/18 (28%) cases, the patient in 11/18 (61%) cases and both in 2/18 (11%). The decision favoring the higher dose arm was made by the patient in most cases 9/12 (75%), both physician and patient in 2/12 (16%) and the physician in only 1/12 (8%) cases. Despite the limitations of lack of randomization, patients' baseline characteristics were well balanced with regard to age, performance status, TRM score, lab values and cytogenetic/mutational risk categories (Table 1). One patient was not yet evaluable for response or TRM at data cutoff. Rates of MRDneg CR were comparable: 6/19 (32%) in the lower and 3/14 (21%) in the higher dose groups (p=0.70). CR+CRi rates were also similar in both arms (43% vs. 56% in lower vs. higher dose arms; p=0.47). Three (16%) patients experienced early death in the lower dose arm vs. 1 (7%) in the higher dose arm (p=0.43). With a median follow up of 4.2 months, there was no survival difference between the two groups (median OS of 6.1 months in the lower vs. 4.7 months in the higher dose arm; p=0.81; Figure 1). Conclusions: Randomization of medically unfit patients to lower- vs. higher-intensity therapy was not feasible, and physicians rarely chose higher intensity therapy in this patient group. Acknowledging the limitation of short follow-up time and small sample size, our trial did not identify significant differences in outcomes between intensive and non-intensive chemotherapy. Analysis of differences in QOL and healthcare resource utilization between groups is ongoing. Disclosures Halpern: Pfizer Pharmaceuticals: Research Funding; Bayer Pharmaceuticals: Research Funding. Othus:Celgene: Other: Data Safety and Monitoring Committee. Gardner:Abbvie: Speakers Bureau. Percival:Genentech: Membership on an entity's Board of Directors or advisory committees; Pfizer Inc.: Research Funding; Nohla Therapeutics: Research Funding. Scott:Incyte: Consultancy; Novartis: Consultancy; Agios: Consultancy; Celgene: Consultancy. Becker:AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; Accordant Health Services/Caremark: Consultancy; The France Foundation: Honoraria. Oehler:Pfizer Inc.: Research Funding; Blueprint Medicines: Consultancy. Walter:BioLineRx: Consultancy; Astellas: Consultancy; Argenx BVBA: Consultancy; BiVictriX: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Seattle Genetics: Research Funding; Race Oncology: Consultancy; Aptevo Therapeutics: Consultancy, Research Funding; Kite Pharma: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding. OffLabel Disclosure: Cladribine is FDA-approved for Hairy Cell Leukemia. Here we describe its use for AML, where is is also widely used with prior publications supporting its use

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The Protein Kinase C Inhibitor MS-553 for the Treatment of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128871 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2077-2077

Author(s):

Elizabeth M. Muhowski ◽

Amy M. Lehman ◽

Sean D. Reiff ◽

Janani Ravikrishnan ◽

Rose Mantel ◽

...

Keyword(s):

Research Funding ◽

Ohio State University ◽

Lymphocytic Leukemia ◽

State University ◽

Advisory Committees ◽

University Patents ◽

Kinase C ◽

Hla Dr ◽

Bcr Signaling ◽

Equity Ownership

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells. Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation. Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p<.01, n=5) , ERK (46%, p=0.02, n=4) , and IκBα (56%, p=0.04, n=5) compared to vehicle treated, stimulated samples. CpG-mediated TLR9 stimulation increases expression of CD86 and HLA-DR in primary CLL cells. In baseline samples from ibrutinib treated patients, 10 µM MS-553 decreased expression of CD86 by 34% and HLA-DR by 91%. In matched patient samples post-relapse due to ibrutinib resistance, MS-553 (10 µM) maintained the ability to decrease expression of CD86 (49%) and HLA-DR (84%). Pro-inflammatory cytokine expression by primary CLL cells stimulated with anti-IgM decreased in the presence of 5 µM MS-553, with CCL3 decreasing by 36% (p=0.06, n=5) and CCL4 decreasing by 79% (p<.01, n=4) compared to vehicle treated, stimulated controls. TNFα expression by healthy T cells increased with anti-CD3 and anti-CD28 stimulation; 1 µM MS-553 reduced TNFα expression by 97% compared to vehicle treated, stimulated controls (p<.01, n=9). Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL. Disclosures Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; 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; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.

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Phase I Study of Ixazomib with Conventional Chemotherapy in the Treatment of Acute Myeloid Leukemia in Older Adults

Blood ◽

10.1182/blood-2020-140584 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-8

Author(s):

Philip C. Amrein ◽

Eyal C. Attar ◽

Geoffrey Fell ◽

Traci M. Blonquist ◽

Andrew M. Brunner ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Remission Rate ◽

Conventional Chemotherapy ◽

Advisory Committees ◽

Secondary Aml ◽

Grade 3 ◽

Acute Myeloid

Introduction: Outcomes for acute myeloid leukemia (AML) among older patients has remained largely unchanged for decades. Long-term survival for patients aged >60 years is poor (median survival 10.5 months). Targeting the proteasome in AML is attractive, since leukemia stem cells have demonstrated sensitivity to proteasome inhibition in preclinical models, perhaps through down regulation of nuclear NF-KB (Guzman, Blood 2001). AML cell lines are susceptible to synergistic cytotoxicity when bortezomib, a proteasome inhibitor, is combined with daunorubicin and cytarabine. We have shown that adding bortezomib to standard treatment in AML results in a high remission rate, although grade 2 sensory neurotoxicity was noted in approximately 12% of treated patients. A newer generation proteasome inhibitor, ixazomib, is less frequently associated with neurotoxicity, and, therefore, was selected for combination with conventional chemotherapy in this phase I trial. The primary objective of this study was to determine the maximum tolerated dose (MTD) of ixazomib in combination with conventional induction and consolidation chemotherapy for AML. Herein are the initial results of this trial. Methods: Adults >60 years of age with newly diagnosed AML were screened for eligibility. Patients with secondary AML were eligible, including those with prior hypomethylating agent therapy for myelodysplastic syndromes (MDS). We excluded those with promyelocytic leukemia. There were 2 phases in this study. In the first phase (A), the induction treatment consisted of the following: cytarabine 100 mg/m2/day by continuous IV infusion, Days 1-7; daunorubicin 60 mg/m2/day IV, Days 1, 2, 3, and ixazomib was provided orally at the cohort dose, Days 2, 5, 9, and 12. Consolidaton or transplant was at the discretion of the treating physician in phase A. In the second phase (B), induction was the same as that with the determined MTD of ixazomib. All patients were to be treated with the following consolidation: cytarabine at 2 g/m2/day, days 1-5 with ixazomib on days 2, 5, 9, and 12 at the cohort dose for consolidation. A standard 3 + 3 patient cohort dose escalation design was used to determine whether the dose of ixazomib could be safely escalated in 3 cohorts (1.5 mg/day, 2.3 mg/day, 3.0 mg/day), initially in induction (phase A) and subsequently in consolidation (phase B). The determined MTD of ixazomib in the first portion (A) of the trial was used during induction in the second portion (B), which sought to determine the MTD for ixazomib during consolidation. Secondary objectives included rate of complete remission, disease-free survival, and overall survival (OS). Results: Thirty-six patients have been enrolled on study, and 28 have completed dose levels A-1 through A-3 and B1 through B-2. Full information on cohort B-3 has not yet been obtained, hence, this report covers the experience with the initial 28 patients, cohorts A-1 through B-2. There were 12 (43%) patients among the 28 with secondary AML, either with prior hematologic malignancy or therapy-related AML. Nineteen patients (68%) were male, and the median age was 68 years (range 61-80 years). There have been no grade 5 toxicities due to study drug. Three patients died early due to leukemia, 2 of which were replaced for assessment of the MTD. Nearly all the grade 3 and 4 toxicities were hematologic (Table). There was 1 DLT (grade 4 platelet count decrease extending beyond Day 42). There has been no grade 3 or 4 neurotoxicity with ixazomib to date. Among the 28 patients in the first 5 cohorts, 22 achieved complete remissions (CR) and 2 achieved CRi, for a composite remission rate (CCR) of 86%. Among the 12 patients with secondary AML 8 achieved CR and 2 achieved CRi, for a CCR of 83%. The median OS for the 28 patients has not been reached (graph). The 18-month OS estimate was 65% [90% CI, 50-85%]. Conclusions: The highest dose level (3 mg) of ixazomib planned for induction in this trial has been reached safely. For consolidation there have been no serious safety issues in the first 2 cohorts with a dose up to 2.3 mg, apart from 1 DLT in the form of delayed platelet count recovery. The recommended phase 2 dose of ixazomib for induction is 3 mg. Accrual to cohort B-3 is ongoing. Notably, to date, no grade 3 or 4 neurotoxicity has been encountered. The remission rate in this older adult population with the addition of ixazomib to standard chemotherapy appears favorable. Figure Disclosures Amrein: Amgen: Research Funding; AstraZeneca: Consultancy, Research Funding; Takeda: Research Funding. Attar:Aprea Therapeutics: Current Employment. Brunner:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Forty-Seven Inc: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Takeda: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Hobbs:Constellation: Honoraria, Research Funding; Novartis: Honoraria; Incyte: Research Funding; Merck: Research Funding; Bayer: Research Funding; Jazz: Honoraria; Celgene/BMS: Honoraria. Neuberg:Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Fathi:Blueprint: Consultancy; Boston Biomedical: Consultancy; BMS/Celgene: Consultancy, Research Funding; Novartis: Consultancy; Kura Oncology: Consultancy; Trillium: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Abbvie: Consultancy; Pfizer: Consultancy; Newlink Genetics: Consultancy; Forty Seven: Consultancy; Trovagene: Consultancy; Kite: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Amphivena: Consultancy; PTC Therapeutics: Consultancy; Agios: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Jazz: Consultancy. OffLabel Disclosure: Ixazomib is FDA approved for multiple myeloma. We are using it in this trial for acute myeloid leukemia.

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The Novel Dihydroorotate Dehydrogenase (DHODH) Inhibitor PTC299 Inhibit De Novo Pyrimidine Synthesis with Broad Anti-Leukemic Activity Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-139940 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Sujan Piya ◽

Marla Weetall ◽

Josephine Sheedy ◽

Balmiki Ray ◽

Huaxian Ma ◽

...

Keyword(s):

Dna Damage ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

De Novo ◽

Dihydroorotate Dehydrogenase ◽

Nucleotide Synthesis ◽

Inhibition Of Proliferation ◽

Acute Myeloid

Introduction: Acute myeloid leukemia (AML) is characterized by both aberrant proliferation and differentiation arrest at hematopoietic progenitor stages 1,2. AML relies upon de novo nucleotide synthesis to meet a dynamic metabolic landscape and to provide a sufficient supply of nucleotides and other macromolecules 3,4. Hence, we hypothesized that inhibition of de novo nucleotide synthesis would lead to depletion of the nucleotide pool and pyrimidine starvation in leukemic cells compared to their non-malignant counterparts and impact proliferative and differentiation inhibition pathways. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Aim: We investigated the pre-clinical activity of PTC299 against AML in primary AML blasts and cytarabine-resistant cell lines. To confirm that PTC299 effects are due to inhibition of de novo pyrimidine nucleotide synthesis for leukemic growth, we specifically tested the impact of uridine and orotate rescue. In addition, a comprehensive analysis of alteration of metabolic signaling in PI3K/AKT pathways, apoptotic signatures and DNA damage responses were analyzed by Mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of DHODH inhibition was confirmed in an AML-PDX model. Results: The IC50s for all tested cell lines (at 3 day) and primary blasts (at 5-7 day) were in a very low nanomolar range: OCI-AML3 -4.43 nM, HL60 -59.7 nM and primary samples -18-90 nM. Treatment of AML in cytarabine-resistant cells demonstrated that PTC299 induced apoptosis, differentiation, and reduced proliferation with corresponding increase in Annexin V and CD14 positive cells (Fig.1). PTC299-induced apoptosis and inhibition of proliferation was rescued by uridine and orotate. To gain more mechanistic insights, we used an immunoblotting and mass cytometry (CyTOF) based approach to analyze changes in apoptotic and cell signaling proteins in OCI-AML3 cells. Apoptotic pathways were induced (cleaved PARP, cleaved Caspase-3) and DNA damage responses (TP53, γH2AX) and the PI3/AKT pathway were downregulated in response to PTC299. In isogenic cell lines, p53-wildtype cells were sustained and an increased DNA damage response with corresponding increase in apoptosis in comparison to p53-deficient cells was shown. (Fig.2) In a PDX mouse model of human AML, PTC299 treatment improved survival compared to mice treated with vehicle (median survival 40 days vs. 30 days, P=0.0002) (Fig.3). This corresponded with a reduction in the bone marrow burden of leukemia with increased expression of differentiation markers in mice treated with PTC299 (Fig.3). Conclusion: PTC299 is a novel dihydroorotate dehydrogenase (DHODH) inhibitor that triggers differentiation, apoptosis and/or inhibition of proliferation in AML and is being tested in a clinical trials for the treatment of acute myeloid malignancies. Reference: 1. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 2017; 129(12): 1577-1585. e-pub ahead of print 2017/02/06; doi: 10.1182/blood-2016-10-696054 2. Quek L, Otto GW, Garnett C, Lhermitte L, Karamitros D, Stoilova B et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 2016; 213(8): 1513-1535. e-pub ahead of print 2016/07/06; doi: 10.1084/jem.20151775 3. Villa E, Ali ES, Sahu U, Ben-Sahra I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 2019; 11(5). e-pub ahead of print 2019/05/22; doi: 10.3390/cancers11050688 4. DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv 2016; 2(5): e1600200. e-pub ahead of print 2016/07/08; doi: 10.1126/sciadv.1600200 Disclosures Weetall: PTC Therapeutic: Current Employment. Sheedy:PTC therapeutics: Current Employment. Ray:PTC Therapeutics Inc.: Current Employment. Konopleva:Genentech: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Ascentage: Research Funding; Agios: Research Funding; Kisoji: Consultancy; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; Calithera: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Sanofi: Research Funding. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Borthakur:BioLine Rx: Consultancy; BioTherix: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; PTC Therapeutics: Research Funding; FTC Therapeutics: Consultancy; Curio Science LLC: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding.

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