Introduction: Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes. Immunosuppressive regimens using methotrexate or cyclophosphamide are utilized as front-line treatments in LGL leukemia but are not curative nor are they effective in all patients. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs.
We have previously reported that STAT3 is constitutively active in LGL leukemia cells and up to 40% of patients harbor activating mutations in STAT3 within the leukemic clone. We have also reported that the pro-survival Bcl-2-family protein Mcl-1 is upregulated in LGL leukemia patients. Additionally, we have identified MCL1 as a transcriptional target of STAT3 in leukemic LGLs. Recently, novel small molecule inhibitors of Mcl-1 have been identified including marinopyrrole A (maritoclax) and its derivative KS18. These inhibitors effectively induced apoptosis in Mcl-1-dependent cells and suppressed the growth of Mcl-1-dependent hematologic cancer xenografts in nude mice without apparent toxicity. Thus, this study further investigates the role of Mcl-1 in LGL leukemia and the potential therapeutic application of the Mcl-1 antagonists maritoclax and KS18.
Results: Knockdown of Mcl-1 in human-derived LGL leukemia cell lines (TL1 and NKL) resulted in decreased cell viability by MTS assay and increased apoptosis by flow cytometry in Mcl-1 siRNA versus scramble siRNA-transfected cells (NKL and TL-1 cells: p < 0.001). Treatment with maritoclax or KS18 decreased cell proliferation and increased apoptosis in NKL, TL1 and RNK16 (rat-derived LGL leukemia cell line) cells (Maritoclax, EC50: 2.45, 1.43 and 1.23 μM respectively) (KS18, EC50: 1.30, 0.60 and 0.31 μM respectively). Ex vivo treatment with maritoclax and KS18 induced selective apoptosis in PBMCs from LGL leukemia patients (NK-LGL, n = 6; T-LGL, n = 12) but not normal donor PBMCs (n = 4) (Maritoclax and KS18, p < 0.0001). Importantly, there was no significant difference in treatment efficacy in patient cells with wild-type STAT3 (n = 9) versus mutant STAT3 (Y640F, n = 5; D661Y, n = 3). We then proceeded to mechanistic studies of Mcl-1 antagonism. Both maritoclax and KS18 induced selective and dose-dependent Mcl-1 degradation by western blot in NKL, TL1, RNK16 and PBMCs from LGL leukemia patients. We further delineated that this effect was proteasome dependent as it is reversible by pre-treatment with MG132, a proteasome inhibitor.
Preclinical in vivo studies were then conducted in Fisher F344 rats that were transplanted with primary leukemic LGL cells and monitored for signs of leukemia through serial CBC and body weight measurements. Leukemic rats developed progressive anemia, thrombocytopenia and leukocytosis and significant weight loss. Rats were treated with vehicle or KS18 (2.5 or 5.0 mg/kg i.p. daily) upon detection of leukemic features. In non-leukemic KS18-treated rats, there were no observed toxicities and there was no difference in survival or weight gain versus vehicle-treated non-leukemic rats. However, leukemic KS18-treated rats exhibited findings consistent with tumor lysis syndrome, including sudden death associated with severely increased serum LDH and increased apoptosis in heart and spleen tissues. At necropsy, leuk/KS18 rats exhibited significantly decreased infiltration and elevated apoptosis of leukemic cells in heart and spleen tissues. Conversely, in leuk/veh rats, spleen tissue lacked normal histologic structure with red pulp replaced almost completely by leukemia cells. Ex vivo KS18 treatment also decreased the viability of PBMC and spleen cells from leukemic animals (p < 0.0001). These data indicate that Mcl-1 is a promising therapeutic target as evidenced by potent therapeutic efficacy of KS18 in LGL cell lines and primary patient material and in an aggressive rat model of LGL leukemia. Further studies are required in order to optimize drug dose, timing and concentrations of KS18 in order to prevent potential tumor lysis syndrome and optimize therapeutic benefit.
Gru: Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.