The Protein Kinase C Inhibitor MS-553 for the Treatment of Chronic Lymphocytic Leukemia
Authors: Muhowski Elizabeth M. , Lehman Amy M. , Reiff Sean D. , Ravikrishnan Janani , Mantel Rose , Misra Shrilekha , Niesman Michael , Zhang Kai , Blachly James S. , Sampath Deepa , Byrd John C. , Woyach Jennifer 
Published: November 2019

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells.
Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation.
Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p<.01, n=5) , ERK (46%, p=0.02, n=4) , and IκBα (56%, p=0.04, n=5) compared to vehicle treated, stimulated samples. CpG-mediated TLR9 stimulation increases expression of CD86 and HLA-DR in primary CLL cells. In baseline samples from ibrutinib treated patients, 10 µM MS-553 decreased expression of CD86 by 34% and HLA-DR by 91%. In matched patient samples post-relapse due to ibrutinib resistance, MS-553 (10 µM) maintained the ability to decrease expression of CD86 (49%) and HLA-DR (84%). Pro-inflammatory cytokine expression by primary CLL cells stimulated with anti-IgM decreased in the presence of 5 µM MS-553, with CCL3 decreasing by 36% (p=0.06, n=5) and CCL4 decreasing by 79% (p<.01, n=4) compared to vehicle treated, stimulated controls. TNFα expression by healthy T cells increased with anti-CD3 and anti-CD28 stimulation; 1 µM MS-553 reduced TNFα expression by 97% compared to vehicle treated, stimulated controls (p<.01, n=9).
Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL.

Disclosures
Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.