MicroRNAs regulate novel signaling pathways targetable by PI3K, MEK, BCL6 and EZH2 Inhibitors in ibrutinib resistance mantle cell lymphoma

Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma. Bruton’s tyrosine kinase (BTK) is a key component of B-cell receptor (BCR) signalling, implicated in B-cell cancers and an effective therapeutic target in MCL. Resistance to the BTK inhibitor ibrutinib is a major clinical challenge that has prompted a search for alternative therapeutic options for this patient population. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by silencing messenger RNA (mRNA) targets. MiRNAs have an important role in cancer development and drug response. We hypothesized that miRNA-mRNA interactions may help identify the molecular mechanism involved in ibrutinib resistance in MCL and may suggest novel therapeutic options for patients with ibrutinib-reistant disease. Methods: We generated 3 ibrutinib-resistant cell lines: Jeko_R, Rec_R and Mino_R by gradually increasing ibrutinib dosage over time. MiRNA and mRNA expression profiles were determined using small-RNA and RNA next-generation sequencing, respectively. MiRNA/gene and protein expression profiles were validated in sensitive and resistant cell lines, in patient-derived xenograft (PDX) models (n = 10) and in patient biopsies (n = 25) by qRT-PCR, Western analyses and immunohistochemistry (IHC). MEK, PI3K, BCL2, BCL6 and EZH2 inhibitors were used to target overexpressed signaling pathways in ibrutinib-resistant cells. Results: Ibrutinib-sensitive cells had different miRNA and mRNA expression signatures compared with those of ibrutinib-resistant cells. Crossing of miRNA binding site sequences (seed region) with their mRNA target sequences revealed potential involvement of miRNAs in regulating pro-cancerous pathways (e.g., MAPK, PI3K-AKT, mTOR, NFkB) and mitochondrial energy-related pathways (Oxphos signaling and the tricarboxylic acid cycle) in ibrutinib-resistant cell lines. A subset of miRNAs (miRs-221, 146a, 182, 342 and the let-7 family members), which regulate the survival pathways MAPK-ERK and PI3K cascade, were downregulated in ibrutinib-resistant cell lines, PDX, and in patients with ibrutinib-resistant MCL. Moreover, MCL-resistant cells overexpressed pERK, pAKT, BCL6 and EZH2 proteins. Treatment of the resistant cells with specific inhibitors such as cobimetinib (MEKi), idelalisib (PI3Ki), FX1 (BCL6i), and tazemetostat (EZH2i) resulted in significant downregulation of these proteins and reduced cell proliferation. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. M. Gomes da Silva Consultant or advisory role: Janssen, Roche, Gilead, Abbvie Research funding: Astrazeneca O. Abdel-Wahab Consultant or advisory role: H3B Biomedicine, Foundation Medicine Inc., Merck, Prelude Therapeutics, Janssen, Envisagenics Inc., AIChemy, Harmonic Discovery Inc., and Pfizer Boulder Research funding: H3B Biomedicine, Nurix Therapeutics, Minovia Therapeutics, and LOXO Oncology.