Abstract
Acute myeloid leukemia (AML) therapy has remained relatively unchanged for more than 40 years with the majority of patients not achieving long-term remission when treated with currently available agents. Novel strategies are urgently needed to improve outcomes. The constitutive dysregulation of protein synthesis/turnover contributes to disease progression and drug resistance in many forms of cancer including AML. p97 (VCP) is a master regulator of protein turnover that has been implicated in oncogenesis and malignant pathogenesis. CB-5083 is a first-in-class selective and potent orally available inhibitor of p97 that in currently being evaluated in phase I clinical trials in patients with multiple myeloma and advanced solid tumors. To assess the potential benefit of p97 inhibition as a novel approach for AML therapy, we investigated the efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of CB-5083 in a panel of human AML cell lines with diverse genetic backgrounds, primary AML specimens from both newly diagnosed and relapsed/refractory patients, and xenograft mouse models of AML. In vitro treatment with CB-5083 potently diminished the viability of AML cell lines (n = 7) and primary CD34+ blasts obtained from patients (n = 10) with IC50s significantly below 1 µM (range 200 - 700 nM) in all lines and specimens evaluated to date. Diminished viability was associated with reduced clonogenic survival and increased apoptosis in AML cell lines and primary blasts. In contrast to many conventional and experimental drugs that are less active against primary AML cells than established AML cell lines, primary cells exhibited sensitivity to CB-5083 that was similar to cell lines. Additionally, CB-5083 was highly active in 3 different cell line models of cytarabine resistance and primary cells from refractory AML patients. This suggests that CB-5083 may be effective for patients who are relapsed/refractory to conventional therapy. In vitro PD analyses demonstrated that CB-5083 rapidly triggered the accumulation of ubiquitin-conjugated proteins, activated the unfolded protein response (UPR), disrupted STAT5 signaling, reduced levels of key STAT5 targets including BCL-xL and PIM-2, and induced apoptosis. The pro-apoptotic effects of CB-5083 were associated with activation of the endoplasmic reticulum (ER) resident initiator caspase-4 and induction of the BH3-only protein NOXA, which has been previously demonstrated to be an important mediator of cell death induced by other agents that disrupt protein homeostasis. RNA sequencing (RNASeq) gene ontology (GO) analyses of MV4-11 and MOLM-13 AML cells following treatment with CB-5083 demonstrated that short-term treatment (6h) caused significant increases in multiple regulators of the unfolded protein response, protein biosynthesis, and other ubiquitin-related pathways (p<0.001). Results were confirmed by qRT-PCR. The in vivo anti-leukemic activity of CB-5083 was investigated in two different xenograft mouse models of AML: the FLT3-ITD+ MV4-11 cell line and APML HL-60 cells. Oral administration of CB-5083 (once daily, 4 days on, 3 days off) was well tolerated and induced disease regression in both xenograft models (p<0.01). In vivo PD studies demonstrated that administration of CB-5083 led to reduced AML cell proliferation (PCNA), to the induction of apoptosis (active caspase-3), and pathway inhibition as evidenced by poly-ubiquitin accumulation and elevated expression of CHOP, GRP78, and NOXA. PK-PD analyses demonstrated a correlation between the kinetics of the in vivo PD effects and drug exposure. Our collective preclinical data demonstrate that p97 inhibition is a very effective novel anti-leukemic strategy and support clinical investigation of CB-5083 in patients with relapsed/refractory AML.
Disclosures
LE Moigne: Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Anderson:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.
At the Crossroads of Survival and Death: The Reactive Oxygen Species–Ethylene–Sugar Triad and the Unfolded Protein Response
