Identification of Novel Synthetic Lethal Partners of NAMPT Inhibitor By CRISPR-Cas9 Screens in Acute Myeloid Leukemia
Authors: Zhang Pu , Brinton Lindsey , Blachly James S. , Byrd John C. , Lapalombella Rosa 
Published: November 2019

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.