Efficacy of Vcp/p97 Inhibitor, CB-5339, Alone and in Combinations Against High-Risk AML, Including Those with Genetic Lesion in TP53

Valosin-containing protein (VCP)/p97 or p97 is an AAA+ type ATPase involved in quality control of cellular proteins in normal and transformed cells, especially under cellular stress. VCP/p97 alleviates proteotoxic stress by facilitating bulk protein degradation through the ubiquitin-proteasome system (UPS), harnessing the energy of ATP hydrolysis to unfold and extract its ubiquitylated protein substrates from protein complexes or organelles and targeting them for proteasomal degradation, e.g., ER-associated degradation (ERAD). Targeted inhibition of p97 induces lethal ER stress. The vital role of p97 in protein homeostasis has been recognized as one of the key non-oncogenic addictions in transformed cells. Targeting p97 has been previously shown to yield preferential lethality in cancer versus normal cells and exhibit in vivo efficacy against xenograft models of cancers. By probing the CRISPR-screen dependency map (DepMap), we found that p97 is a dependency across AML cell lines, including those harboring alterations in genes that indicate an increased risk or poor prognosis. CB-5339 is a selective and potent, ATP-competitive inhibitor of p97, with only low inhibitory activity against other ATPases, AAA+ ATPases, and kinases. CB-5339 was previously shown to induce accumulation of polyubiquitylated proteins, retention of ERAD substrates, and lethal ER stress in cancer cells mediated by CHOP, DR5 and NOXA. In present studies, we determined that treatment with CB-5339 dose-dependently (200 to 1000 nM for 48 hours) induces in vitro loss of viability in AML cell lines (OCI-AML3, MOLM13, SET2 and HEL92.1.7 cells), as well as in fresh, patient-derived (PD) AML cells, including those with TP53 mutations and/or TP53 allelic loss, or with 3q26 (MECOM locus) lesions and EVI1 overexpression. Utilizing MOLM13 cells edited to express the missense mutation R175H or R248Q, or the null allele of TP53, introduced via CRISPR/Cas9 into the endogenous TP53 locus, we determined that CB-5339 was active against MOLM13 cells (with MLL-AF9, FLT3-ITD and wild-type TP53) and retained significant activity against MOLM13 cells with TP53-R175H, TP53-R248Q and MOLM13-TP53^-/-. Comparing ATAC-Seq and RNA-Seq findings in CB-5339-treated versus -untreated MOLM13 cells we found concordant, significantly altered chromatin accessibility and mRNA expressions of gene-loci, e.g., increase in ATF4, ATF3, HSPA5 (GRP78), NOXA, BBC3 (PUMA), FBXO32, ITGAM, and CD86, but decrease in FOXA1, TLR4, CLEC12A, CXCR4 and IL7R. Following CB-5339 treatment, gene-set enrichment analyses (GSEA) showed significantly increased normalized enrichment scores of the gene-set involved in ER stress response, not only in MOLM13 with WT-TP53 but also in those with the TP53 mutations or allelic loss of TP53. However, treatment with CB-5339 increased levels of p-ATM, p-TP53 and TP53 only in MOLM13 and OCI-AML3 (each expressing only WT-TP53), without altering the protein levels of MDM2/4. In contrast, CB-5339-induced loss of viability correlated with augmented levels of ATF4, CHOP, as well as of pro-death proteins NOXA, DR5 and active BAX in AML cells regardless of TP53 status. Importantly, similar effects were also noted in PD AML cells with or without TP53 mutation. Co-treatment with relatively lower concentrations of CB-5339 (100 to 225 nM) and venetoclax (5 to 20 nM) exerted synergistic in vitro lethality in not only MOLM13 but also PD AML cells with WT-TP53, as well as in SET2 and PD AML cells harboring mutant TP53. Co-treatment with CB-5339 and pan-BET inhibitor OTX015 was also synergistically lethal against MOLM13 and PD AML cells with or without mutant TP53. Finally, in a tail-vein infused, luciferase transduced, aggressive xenograft model of MOLM13 cells, after AML engraftment, co-treatment for 3 weeks with CB-5339 (50 mg/kg/day, PO) and either venetoclax (30 mg/kg/day, PO) or OTX015 (30 mg/kg/day, PO), as compared to treatment with vehicle or each drug alone, significantly reduced the AML burden and improved median and overall survival of the NSG mice, without inducing significant toxicity. Taken together, these findings highlight that CB-5339 induces lethal ER stress in AML cells regardless of the TP53 status, and underscore the promise of CB-5339 treatment alone and in rational combinations in exerting efficacy against AML, including those with high-risk genetic alterations in TP53 or chromosome 3q26 lesions and EVI1 overexpression.

Kadia:Glycomimetics: Research Funding; Astex: Honoraria; Iterion: Research Funding; Ascentage: Research Funding; cyclacel: Research Funding; Delta-Fly: Research Funding; Servier: Consultancy; cellenkos: Research Funding; AstraZeneca: Research Funding; PinotBio: Consultancy; JAZZ: Consultancy, Research Funding; Regeneron: Research Funding; Novartis: Consultancy; Pfizer: Research Funding; Amgen: Research Funding; Agios: Consultancy; Astellas: Research Funding; Genfleet: Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Daver:Agios, Celgene, SOBI and STAR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos and Jazz Pharmaceuticals: Other: Data monitoring committee member; Karyopham Therapeutics and Newave Pharmaceutical: Research Funding; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Novartis, Jazz, Amgen, Servier, Karyopharm, Trovagene, Trillium, Syndax, Gilead, Pfizer, Bristol Myers Squibb, Kite, Actinium, Arog, Immunogen, Arcellx, and Shattuck: Consultancy, Other: Advisory Role; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Gilead, Immunogen, Pfizer, Bristol Myers Squibb, Trovagene, Servier, Novimmune, Incyte, Hanmi, Fate, Amgen, Kite, Novartis, Astex, KAHR, Shattuck, Sobi, Glycomimetics, Trillium: Research Funding. Sasaki:Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Takahashi:Celgene/BMS: Consultancy; GSK: Consultancy; Agios: Consultancy; Ostuka Pharmaceuticals: Honoraria; Novartis: Consultancy; Symbio Pharmaceuticals: Consultancy; Mission Bio: Honoraria; Illumina: Honoraria. Loghavi:Abbvie: Consultancy, Current equity holder in publicly-traded company; QualWorld: Consultancy; Amgen: Research Funding; PeerView: Honoraria; Astellas: Research Funding; GLG: Consultancy. DiNardo:Cleave: Research Funding; Astex: Research Funding; AbbVie: Consultancy, Research Funding; Jazz: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Kura: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Bluebird Bio: Honoraria; Gilead: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Foghorn: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Novartis: Honoraria; LOXO: 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; Forma: Research Funding; GenMab: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Research Funding. Vargas:Cleave Therapeutics: Current Employment.