Elucidating Mechanisms of Therapy-Resistance to Interferon-alfa in Myeloproliferative Neoplasm Stem Cells

点击翻译按钮获取中文摘要

PROJECT SUMMARY Recombinant interferon-alpha (IFN) remains a highly effective therapy for patients with myeloproliferative neoplasms (MPN). We recently identified that patients with CALR-mutated MPN frequently exhibit normalization of blood counts (i.e. clinical response), but often do not exhibit a decrease in tumor burden (i.e. molecular response), providing an informative model to decipher the mechanisms of therapy-resistance to IFN. Interrogating the molecular impact of IFN on human MPN stem cells may reveal critical insights into mechanisms of therapy-resistance. Thus, we applied our innovative Genotyping of Transcriptomes (GoT) platform – that captures the mutation status and single-cell whole transcriptomes (scRNA-seq) within the same cells – CD34+ cells from serial bone marrow (BM) aspirates from patients with CALR-mutated MPN treated with IFN. Strikingly, we observed that IFN caused major shifts in the differentiation landscapes, distinctly in the mutated and wildtype progenitors: IFN exposure on wildtype cells resulted in a large expansion of lymphoid progenitors, while the mutated cells, in contrast, displayed an expansion of the granulo-monocytic (GM) progenitors (with a less striking expansion of the lymphoid compartment). Our preliminary data indicate that (1) the GM differentiation bias of CALR-mutated stem cells may underlie therapy-resistance, and that (2) the CALR-mutation induced UPR may prime the mutated stem cells toward the GM lineage and play a role in therapy-resistance. To interrogate these hypotheses, we will determine the transcription factor (TF) networks that govern the IFN-induced differentiation shifts by applying a novel single-cell multi-omics platform that captures RNA-seq, chromatin accessibility and somatic genotyping within the same thousands of single cells (GoT-ATAC) to the same IFN-treated cohort (Aim 1a), and by targeting these TF networks in mouse models (Aim 1b). We will define the role of UPR in therapy- resistance in treated CALR-mutated cells through GoT-ATAC and chromatin binding assays (Aim 2a) and by assessing perturbations to the UPR pathways in mouse models (Aim 2b). Finally, we will determine the impact of co-mutations in DNMT3A or ASXL1 in therapy-resistance to IFN in CALR-mutated MPN via application of single-cell multi-omics platforms to clinical samples (Aim 3a) and interrogation of IFN effects on novel mouse models with double mutations (Aim 3b). The project is centered on a conceptually innovative framework in which we superimpose neoplastic and normal hematopoietic development within the same individuals to define how therapy reshapes differentiation topographies, as a function of mutation status and cell identity. This conceptual innovation is enabled by technical innovations in single-cell multi-omics platforms applied to compelling clinical cohorts, coupled with functional assessments in novel mouse models. These studies have the potential to uncover new insights into the mechanisms of molecular resistance to IFN in MPN, resulting in novel therapeutic approaches.