In Vivo Ablation of NFκB Cascade Effectors Alleviates Disease Burden in Myeloproliferative Neoplasms

Abstract

ABAL and TK equal contributors Multilevel regulation of NFκB activation exists in response to microenvironmental cues. Aberrant hyperactivation of the NFκB cascade propagates oncogenic signaling and inflammation, which together augment disease burden in myeloproliferative neoplasms (MPNs). Here, we systematically ablated NFκB signaling effectors to identify core dependencies using a series of primary samples and syngeneic and patient-derived xenograft (PDX) mouse models. We first examined the impact of Rela abrogation in the Jak2 V617F knock-in mouse model that exemplifies polycythemia vera hallmarks and generated Jak2V617FRela flox/flox Ubc-Cre-ERT2 ( Jak2- Rela fl) mice. Evident suppression of leukocytosis was observed within a week of Rela excision and sustained until endpoint. Elevated hematocrit, monocytes, and CD45.2 myeloid cells were suppressed in both the peripheral blood (PB) and bone marrow (BM) and Jak2- Rela flmice had significant reduction of splenomegaly. We then adopted a second MPN model, where MPL W515L retroviral expression produces a phenotype resembling myelofibrosis. Consistent reduction of pathologic WBC counts, monocytes, and PB and BM CD45.2 myeloid cells were observed in MPL- Rela fl mice. Rela ablation also reduced hematocrit, platelet counts, and CD45.2 GFP+ cells. Importantly, we observed a significant attenuation of disease hallmarks with a decrease of spleen weight and dysmegakaryopoiesis, enhanced survival, and reduction of BM reticulin deposition. We next established PDX models where CD34+ cells from MF patients and healthy BM donors (NBM) were transduced with an sgRNA targeting RELA and engrafted into NSGS mice. Across two MPN PDXs, there was significant suppression of leukemic cells and improvement of disease burden, reduced spleen weights and prolonged survival, and subsequent BM histology revealing improved sclerosis, fibrosis, and dysmegakaryopoiesis. In contrast, NSGS mice engrafted with NBM CD34+ cells did not yield disease symptoms, nor did RELA targeting in healthy cells affect hematopoiesis or measured disease hallmarks. RNA-seq on CD34+ cells from four MPN patients following RELA abrogation revealed augmented and altered expression of genes that are 1) regulators of survival and proliferative pathways: MDM2, TP53INP1, and FAS 2) major ferroptosis mediator GPX4, and 3) cell cycle effectors: CDKN1A (p21), E2F4, and CHEK2. In further investigation of NFκB pathway effectors, we genetically targeted upstream effector Myd88 and transduced Kit+ cells from Myd88-null mice with MPL W515L. Consistent with Rela-knockout, Myd88-deficiency also diminished pathogenic WBC and monocyte count upsurgence, inhibited the accumulation of BM CD45.2 myeloid cells, and reduced spleen weights. The rapid lethality governed by mutant MPL was also delayed upon Myd88 loss, consistent with reduced pathology in spleen and BM sections. While Myd88 and Rela activate NFκB, miR-146a is repressive. We performed further knock-out of MIR146A using PDX models and transplanted MIR146A-KO CD34+ cells into NSGS mice and observed enhanced leukemic engraftment, increased spleen weights, earlier lethality, and evidence of BM sclerosis or fibrosis across both MF PDXs. Importantly, these disease parameters remained unchanged in a NBM PDX following MIR146A-KO. Lastly, in our WashU Cohort of RNA-sequencing performed on 90 CD34+ samples encompassing the spectrum of MPN and secondary acute myeloid leukemia (AML) with NBM controls, we observed significant upregulation of IRAK4 and IL1B in MF and sAML compared to NBM, and their significant expression correlation in MPN. We then pharmacologically inhibited IRAK4 using a small molecule, CA-4948, which is currently under evaluation in phase 1/2a trials of relapsed/refractory AML/myelodysplastic syndrome. In PDX experiments, CA-4948 treatment effectively reduced leukemic engraftment and spleen weights of two MF models without inducing toxicity in a NBM PDX. Through qRT-PCR and mass cytometry, we demonstrate that CA-4948 treatment dampened inflammatory cytokine production induced by IL-1β in primary MPN CD14+ monocytes. Overall, we demonstrate that targeting mediators including Rela, Myd88, and IRAK4 specifically alleviated MPN disease burden without toxicity to healthy tissue and further establish CA-4948 as a promising therapeutic avenue for the treatment of MPN.