Pirtobrutinib (LOXO-305) Is Active and Overcomes ERK Related Pro-Survival Signaling in Ibrutinib Resistant, BTK Cys481 Mutant Expressing WM and ABC DLBCL Lymphoma Cells Driven By Activating MYD88 Mutations
Authors: Munshi Manit , Liu Xia , Kofides Amanda , Tsakmaklis Nickolas , Gustine Joshua , Sarosiek Shayna , Flynn Catherine A , Meid Kirsten , White Timothy P , Leventoff Carly , Patterson Christopher J , Branagan Andrew R. , Gokhale Prafulla , Poitras Michael J , Hunter Zachary R , Guerrera Maria Luisa , Castillo Jorge J. , Yang Guang , Treon Steven P 
Published: November 2021

Abstract
MYD88 mutations are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of Diffuse Large B-cell Lymphoma (ABC DLBCL). Mutated MYD88 activates BTK, and triggers downstream pro-survival signaling that includes NF-kB and ERK (Yang et al, Blood 2013; Blood 2016). ERK related signaling triggers inflammatory cytokine production including IL-6 and IL-10 (Chen et al, Blood 2016). Ibrutinib covalently binds to BTK Cys481 and inactivates BTK and downstream NF-kB and ERK signaling. Ibrutinib is approved for the treatment of WM and is associated with high overall response rates (&gt;90%) and long term progression free survival in WM though intolerance to therapy, as well as resistance related to acquired BTK Cys481 mutations frequently leads to treatment discontinuation. We therefore investigated a novel, non-covalent BTK-inhibitor, pirtobrutinib that binds to BTK at non-Cys481 amino acids (G473-K483). Pirtobrutinib showed highly selective anti-proliferative activity against MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8 and HBL-1) versus MYD88 wild-type (OCI-Ly7, OCI-Ly19, Ramos, and RPMI-8226) cells, with marked apoptotic effect exhibited against primary MYD88 mutated WM cells at pharmacologically achievable levels (100-500 nM). Importantly, pirtobrutinib blocked BTK activity and overcame ibrutinib resistance in BCWM.1 WM and TMD-8 ABC DLBCL cells transduced to express both wild-type and mutated BTK (BTK Cys481Ser) with similar efficacy. The downstream signaling consequences of pirtobrutinib in vector only, wild-type and mutant BTK Cys481 expressing BCWM.1, MWCL-1, TMD-8 and HBL-1 cells was also examined. Treatment of vector only and wild-type BTK Cys481 expressing WM and ABC DLBCL cells with ibrutinib or pirtobrutinib abrogated both p-BTK and p-ERK signaling. In contrast, only pirtobrutinib blocked p-BTK and p-ERK signaling in mutant BTK Cys481 expressing WM and ABC DLBCL cells. In previous studies, we showed that inflammatory cytokine production that included IL-6 and IL-10 driven by ERK triggered ibrutinib resistance in wild-type BTK Cys481 MYD88 mutated lymphoma cells co-cultured with their mutated BTK expressing counterparts (Chen et al, BLOOD 2018). ERK-driven cytokine resistance to ibrutinib was postulated to explain how disease progression occurs in patients with modest variant expression of mutated BTK Cys481 (Woyach et al, JCO 2017; Xu et al, Blood 2017). Co-culture of BTK Cys481 mutated expressing TMD-8 cells with wild-type BTK expressing TMD-8 cells triggered resistance of the latter to ibrutinib. Treatment with pirtobrutinib blocked IL-6 and IL-10 production and overcame the protective effects conferred by BTK Cys481 mutated TMD-8 cells in these experiments. Lastly, oral administration of pirtobrutinib blocked p-BTK and p-ERK in BTK Cys481 mutated TMD-8 tumors xenografted in mice. Our findings therefore show that pirtobrutinib inhibits growth of MYD88 mutated lymphoma cells in a highly selective manner and can trigger apoptosis of primary WM patient BM lymphoplasmacytic cells at levels comparable to ibrutinib. Moreover, pirtobrutinib effectively blocked mutated BTK Cys481 driven BTK and ERK1/2 activation and produced similar cellular efficacy in both BTK wild-type and BTK Cys481 mutated cells. Pirtobrutinib also blocked the protective effect conferred to BTK wild-type cells through paracrine cytokines released by BTK Cys481Ser expressing cells. Lastly, pirtobrutinib blocked BTK and ERK1/2 activation in TMD8-BTK Cys481Ser xenografted mice. The findings support the development of pirtobrutinib in MYD88 driven lymphomas, including those resistant to ibrutinib on the bases of BTK Cys481 mutations.

Disclosures
Branagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.