Newer-Generation HSP90 Inhibitors Have Potent Activity Against Human Mantle Cell Lymphoma in Vitro and in Vivo

Abstract 1651

Mantle cell lymphoma (MCL) is associated with particularly poor outcome, with long-term survival achieved in less than 40% of patients. In addition to the characteristic t(11;14) that results in overexpression of cyclin D1, a variety of other molecular pathways are dysregulated in MCL. Cyclin D1 is a known client of heat shock protein 90 (HSP90), suggesting that inhibitors of HSP90 may destabilize cyclin D1 and have activity in this disease. Yet, first-generation HSP90 inhibitors such as 17-AAG generally lack potency in MCL cell lines. We assessed the pre-clinical activity of second- (NVP-AUY922, PU-H71) and third-generation (NVP-HSP990) HSP90 inhibitors, which have greater potency and superior in vivo pharmacokinetics, in the MCL cell lines Granta519, JeKo1, MAVER1, Rec1, and Z-138. To define the genetics of these lines, we utilized an exon-capture followed by next-generation sequencing approach to identify single nucleotide variants and insertions/deletions across the entire coding sequence of 197 genes known to be recurrently altered in lymphoid malignancies. Sequencing to a median depth of coverage∼400 recovered alterations previously described in MCL (e.g. in ATM, RB1, TP53, NOTCH1) as well as variants in genes that have not previously been associated with MCL (e.g. in MLL2, KDM6A, FLT3, IKZF3, JAK3, RFXAP). Dose response curves of these cell lines treated with structurally diverse HSP90 inhibitors showed 10–100-fold greater potency for NVP-AUY922 (IC₅₀, 3–11 nM), NVP-HSP990 (IC₅₀, 5–24 nM) and PU-H71 (IC₅₀, 40–287 nM), compared with 17-AAG (IC₅₀, 29–1503 nM). In vitro exposure of all lines to 50 nM AUY922 resulted in G0/G1 cell cycle arrest within 6–8 hrs followed by apoptosis within 24–72 hours. Immunoblotting after exposure to AUY922 demonstrated rapid reductions in HSP90 client proteins, including cyclin D1, CDK4 and AKT, in all lines as well as accumulation of HSP70 in all lines except REC1, which harbors an HSP70 locus deletion. Cell killing by AUY922 (based on Annexin V/PI flow cytometry, caspase 3/7 activation and PARP cleavage) varied between cell lines, with Granta519 being the most sensitive (>50% cell death after 24 hr exposure) and Rec1 being the least sensitive (<15% cell death under the same conditions). Co-culture of Granta519, JeKo1, and Z-138 cells with bone marrow stroma had no effect on killing by AUY922, suggesting that HSP90 inhibition may overcome cell non-autonomous pathways that support resistance to other antineoplastic agents. To build on these findings in vivo, we xenografted luciferized MAVER1 (harbors TP53 D281E and JAK3 V722I mutations) and Z-138 (TP53 and JAK3 wild-type) cells into SCID beige mice (10 million cells per mouse). Upon evidence of measurable engraftment, mice (10 per arm) were randomized to receive either AUY922 (50 mg/kg by tail vein injection thrice weekly) or vehicle. Tumors were analyzed from sentinel mice that were sacrificed after 5 days of treatment. Tumors from mice receiving AUY922 had complete loss of cyclin D1 and Ki67 staining by immunohistochemistry. 18F-FLT PET scanning performed on mice xenografted with Z-138 cells demonstrated ∼75% reduction in activity after 5 days of AUY922 treatment. Consistent with these findings, tumor growth was significantly slowed among AUY922-treated animals for both lines, which translated into a survival advantage (p<0.01 for MAVER1 and p=0.03 for Z-138). Finally, in an effort to enhance cell killing, we combined AUY922 with compounds in clinical use for MCL. In JeKo1, MAVER1, Rec1 and Z-138 cells, combinations with AUY922 were either antagonistic (with cytarabine or doxorubicin) or lacked synergistic effects (with bortezomib). AUY922 also failed to block the accumulation of MCL1 induced by exposure to bortezomib. Thus, appropriate drug combination partners for AUY922 in MCL remain to be determined. In conclusion, newer-generation HSP90 inhibitors such as AUY922 have significant single-agent activity across a genetically diverse spectrum of MCLs, can target cyclin D1, CDK4, AKT and other drivers of malignant phenotype, and warrant evaluation in clinical trials.