1004 - The PKCβ inhibitor MS-553 displays preclinical efficacy in BTK inhibitor resistant Chronic Lymphocytic Leukemia

Background: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized through usage of targeted therapies against the B-cell receptor (BCR) signaling cascade, specifically BTK. However, resistance to BTK inhibition has occurred through acquisition of various mutations in BTK or its immediate downstream signaling partner, PLCγ2 (Woyach et al., 2014; Wang et al., 2022). Clinical outcomes for these patients are quite poor, emphasizing the need for alternative therapeutics. Despite the presence of these mutations, BCR signaling remains intact, suggesting that targeting molecules downstream of BTK may be effective. Protein kinase C-β (PKCβ) is a downstream component of the BCR pathway that has been shown to be over-expressed in CLL. Moreover, PKCβ has been shown to be essential to the development of CLL in the Eμ-TCL1 mouse model and its expression in stromal cells to be required for the survival of leukemic B-cells (Holler et al., 2009; Lutzny et al., 2013: El-Gamal et. al., 2014). This suggests that signaling through PKCβ, in both CLL cells and cells in the microenvironment, is essential to promote leukemogenesis. MS-553 is a potent, ATP competitive, reversible inhibitor of multiple PKC isoforms including PKCβ.

Methods: Primary CLL cells were isolated by negative selection and treated with up to 5 μM MS-553. Cell viability was assessed via Annexin V/PI with and without HS-5 stromal cell co-culture. BCR and canonical WNT signaling changes were evaluated by change in target protein phosphorylation or total protein by immunoblot following drugging. Changes in downstream NFκB and WNT signaling were interrogated by RT-qPCR. Using genetically modified TMD8 cells that express only C481S or T474I mutated BTK, we determined MS-553’s effect on cell-viability and BCR signaling. In vivo survival studies were performed using the Eμ-MTCP1 mouse model, which is an aggressive model of CLL, with subsequent randomization into vehicle or MS-553 treatment arms (Walker et al., 2021).

Results: Following 72 hours of drugging, 5 μM MS-553 displayed modest cytotoxicity against primary CLL cells both alone (p < 0.0001, n=8) and in the presence of HS-5 stromal cells (p=0.0008, n=8). At 24 hours, 5 μM MS-553 inhibited downstream BCR and WNT signaling in primary CLL cells, demonstrated by reduced phosphorylation of PKC-β (p=0.0024, n=15) and several of its downstream targets including pGSK3β (p < 0.0001, n=15), pIκBα (p < 0.0001, n=15), and pERK (p=0.0301, n=15), and reduced total protein of β-Catenin (p < 0.0001, n=9), Cyclin D2 (p=0.0375, n=9), and cMyc (p=0.0031, n=9) compared to vehicle treated, stimulated samples. In patient samples, mRNA expression of WNT pathway targets BCL-2, OCT-2, BFL-1, MYC, CD40, Cyclin D1, and Cyclin D2 were all decreased after 48 hours of treatment with 1 or 5 μM MS-553. MS-553 also showed efficacy in the presence of BTK mutations: viability in TMD8 cells was reduced following 72-hour drugging with MS-553 regardless of BTK mutational status (p < 0.05) (Figure 1A: *, 0.05 ≥ p ≥ 0.01: **, 0.01 ≥ p ≥ 0.001). Inflammatory cytokine production by CLL cells is inhibited by MS-553 through its ability to decrease CCL3 and CCL4 cytokine expression in WT and mutant BTK-harboring TMD8 cells (p < 0.001, n=3). As proof of concept, we examined MS-553’s efficacy in vivo, and found a significant reduction in disease progression and increase in survival (median 117 days) when compared to vehicle control (median 56 days, p < 0.0001) (Figure 1B).

Conclusion: Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL, including BTK inhibitor resistant disease. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al., 2022).