Newer-Generation HSP90 Inhibitors Can Overcome Ibrutinib Resistance and Suppress Proliferation in Human Mantle Cell Lymphoma in Vitro and in Vivo

Mantle cell lymphoma (MCL) makes up approximately 6% of non-Hodgkin’s lymphoma cases and has an aggressive disease course with a particularly poor outcome. To date there is no standard treatment and despite good initial response to chemotherapy, most patients relapse with long-term survival achieved in less than 40%. In almost all cases, MCL is distinguished by overexpression of Cyclin D1 (CCND1) usually caused by a translocation between chromosomes 11 and 14. Both CCND1 and BTK are known clients of heat shock protein 90 (HSP90). In previous studies, we demonstrated potent cytotoxic activity of second- (NVP-AUY922, PU-H71) and third-generation (NVP-HSP990) HSP90 inhibitors against the MCL cell lines Granta519, JeKo1, MAVER1, Rec1, and Z-138 (IC50s for AUY922, 3-11nM; HSP990, 5-24nM; PU-H71, 40-287 nM). Immunobloting of MCL cell lines exposed to 50nM AUY922 demonstrated reduced expression of client proteins CCND1, CDK4 and AKT and induction of G0/G1 cell cycle arrest within 6-8h followed by apoptosis within 27h. We now demonstrate that combining Ibrutinib with AU922 results in synergistic or additive effects in all lines except for the highly Ibrutinib- sensitive JEKO-1. In fact, AUY922 overcame ibrutinib-resistance due to activation of non B-cell receptor (BCR)-driven alternative NFkB signaling in Granta519 and Z-138 cells. Due to the promising results of AUY922 we conducted two in vivo studies. Current mouse MCL models are generated by injecting MCL cell lines in matrigel subcutaneously. The MCL grows as a solid tumor and treatment is typically initiated when mean tumor volume is approximately 200 mm³. First, we xenografted luciferized MAVER1 (ibrutinib-sensitive) and Z-138 (ibrutinib-resistant) cells into SCID beige mice (10 million cells per mouse). Mice were randomized to receive either AUY922 (50 mg/kg by tail vein injection thrice weekly) or vehicle. Tumors analyzed from mice sacrificed after 5 days of treatment showed complete loss of cyclin D1 and Ki67 staining by immunohistochemistry in those receiving AUY922. Tumor growth was significantly slowed in AUY922 treated animals in both lines, which translated into a survival advantage (p<0.01) for both lines. To advance beyond xenografting of cell lines, we generated a MCL patient-derived xenograft (PDX) model. Peripheral blood from a patient with relapsed MCL and peripheral blood involvement was xenografted by tail-vein injection. Within 28 days, fatal MCL developed in the spleen and bone marrow as evidenced by flow cytometry, but was also seen in liver and intestines. Xenografted splenocytes were secondarily transplanted into NSG mice. Eighteen days after injection, mice were randomized to receive either AUY922 (50 mg/kg by tail vein injection thrice weekly) or vehicle. After the third dose, mice underwent [18F]-FLT PET imaging. Vehicle mice had PET-avid disease diffusely within lymph nodes, spleen, bone marrow and intestine. Mice treated with 5 days of AUY922 had nearly complete resolution of PET activity in lymph nodes, spleen and bone marrow, as well as approximately 50% reduction in the intestines. Mice were euthanized and immunobloting of the spleens showed pronounced downregulation of both CCND1 and BTK in AUY922-treated mice compared with vehicle. 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, and BTK, and may overcome Ibrutinib resistance.