B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL.

We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

Jain:CareDX: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; AbbVie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Dialectic Therapeutics: Research Funding; Precision Biosciences: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Cellectis: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Incyte: Research Funding; Aprea Therapeutics: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Novalgen: Research Funding; Newave: Research Funding; TransThera Sciences: Research Funding; Ipsen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; ADC Therapeutics: Research Funding; BMS: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Loxo Oncology: Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Genentech: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Medisix: Research Funding; Mingsight: Research Funding; Fate Therapeutics: Research Funding; Servier: Research Funding; MEI Pharma: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Pharmacyclics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Beigene: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses. Wierda:Janssens Biotech Inc: Research Funding; Sunesis: Research Funding; Accutar Biotechnology: Research Funding; Janssens Biotech: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; NIH P30 CA016672/MDACC Cancer Center Support Grant: Research Funding; Miragen: Research Funding; Numab THerapeutics: Research Funding; Pharmacyclics LLC: Research Funding; AstraZeneca/Acerta Pharma: Consultancy, Research Funding; Cyclacel: Consultancy, Research Funding; Loxo Oncology, Inc./Lilly: Research Funding; GlaxoSmithKline: Research Funding; National Comprehensive Cancer Network: Other: Nonrelevant Financial Relationship/Chair, CLL). Supported by the NIH/NCI under award number P30 CA016672 and used MDACC Cancer Center Support Grant (CCSG) shared resources; Bristol Myers Squibb (Juno & Celgene): Consultancy, Research Funding; Gilead Sciences: Research Funding; Genentech: Research Funding; KITE Pharma: Research Funding; Juno Therapeutics: Research Funding; AbbVie: Consultancy, Research Funding; GSK/Novartis: Research Funding; Nurix THerapeutics: Research Funding. Zhang:MingSight Pharmaceuticals: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Niesman:MingSight Pharmaceuticals: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Blachly:Leukemia Diagnostic Device: Patents & Royalties: Being prosecuted; Epigenetic classification of leukemia: Patents & Royalties: PCT conversion filed; AbbVie: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy. Woyach:Newave: Consultancy; Loxo: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy; Abbvie: Consultancy; Schrodinger: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding.

Figure 1
Figure 1
View largeDownload slide
This content is only available as a PDF.
2023
Sign in via your Institution