BCR-ABL1-positive acute lymphoblastic leukemia (ALL) cell survival is dependent on the inositol-requiring enzyme 1 alpha (IRE1α) branch of the unfolded protein response. In the current study, we have focused on exploring the efficacy of a simultaneous pharmacological inhibition of BCR-ABL1 and IRE1α in Philadelphia-positive (Ph+) ALL using tyrosine kinase inhibitor (TKI) nilotinib and the IRE1α inhibitor MKC-8866. The combination of 0.5 µM nilotinib and 30 µM MKC-8866 in Ph+ ALL cell lines led to a synergistic effect on cell viability. To mimic this dual inhibition on a genetic level, pre-B-cells from conditional Xbp1+/fl mice were transduced with a BCR-ABL1 construct and with either tamoxifen-inducible cre or empty vector. Cells showed a significant sensitization to the effect of TKIs after the induction of the heterozygous deletion. Finally, we performed a phosphoproteomic analysis on Ph+ ALL cell lines treated with the combination of nilotinib and MKC-8866 to identify potential targets involved in their synergistic effect. An enhanced activation of p38 mitogen-activated protein kinase α (p38α MAPK) was identified. In line with this findings, p38 MAPK and, another important endoplasmic reticulum-stress-related kinase, c-Jun N-terminal kinase (JNK) were found to mediate the potentiated cytotoxic effect induced by the combination of MKC-8866 and nilotinib since the targeting of p38 MAPK with its specific inhibitor BIRB-796 or JNK with JNK-in-8 hindered the synergistic effect observed upon treatment with nilotinib and MKC-8866. In conclusion, the identified combined action of nilotinib and MKC-8866 might represent a successful therapeutic strategy in high-risk Ph+ ALL.
Abstract Background: Interferon alpha (IFNa) is approved for the therapy of patients (pts) with polycythemia vera (PV), a subtype of myeloproliferative neoplasms (MPN), achieving high hematologic response (HR) rates. A fraction of pts also achieve molecular responses (MR), but clonal factors sensitizing for MR remain elusive. Methods: We integrated colony formation and differentiation assays with single-cell RNA sequencing (scRNAseq) and cell genotyping in PV patient-derived cells vs. healthy controls (HC) to dissect how IFNa targets diseased clones during erythroid differentiation. Results: IFNa significantly decreased colony growth in MPN and HC. Differences in MR were observed, with a higher degree of MR in pts with high variant allele frequency (VAF). While lower STAT1 but higher IFIT2 inducibility of PV vs. HC was observed, responses in individual colonies were much more variable. scRNAseq of colonies demonstrated more mature erythroid PV-derived colonies compared with HC and showed differential activation of JAK-STAT and hypoxia signaling. JAK2V617F-mutant cells showed significantly upregulated STAT5A-, heme- and G2M checkpoint-associated pathways relative to JAK2WT cells from the same pts. IFNa induced viral response genes in mutated cells from PV pts and antagonized the upregulation of metabolism genes. Subgroup analysis revealed that IFNa significantly decreased immature erythrocytic cells in PV (basophilic erythroblasts p <0.05; polychromatic erythroblasts p <0.05) but not in HC. CD71neg‑CD235a+ cells from HC ( p <0.05) but not from PV pts were inhibited by IFNa, and the number of reticulocytes was less affected in PV than in HC. Robust IFNa responses persisted throughout differentiation, with PV showing more pronounced effects than HC. Apoptosis was significantly upregulated by IFNa in PV but not HC, and the most apoptotic cells were characterized by downregulation of ribosomal genes. This link between apoptosis and ribosomal genes was corroborated through analysis of mitochondrial variants, demonstrating IFNa-induced eradication of specific clones, which harbored significantly elevated expression of ribosomal genes. Conclusion: Our findings indicate that PV-derived clones either undergo apoptosis or pass through their typical differentiation cycle, contributing to the long-term exhaustion of mutant cells. The pivotal roles of ribosomal genes and clonal prerequisites in the therapeutic mechanism of IFNa are underscored by our study.
