Abstract
Abstract
Chronic myeloid leukemia (CML) is caused by the oncogenic tyrosine kinase BCR-ABL1, which is uniquely present in the leukemic cells and drives progression of the disease. Small-molecule ABL1 tyrosine kinase inhibitors (TKIs) have transformed prognosis for patients as compared to interferon or standard chemotherapy, with the vast majority of patients achieving deep, long-term remission on treatment (Druker et al., Nature Medicine 2009). However, results from multiple TKI discontinuation studies over the past decade suggest approximately 50-60% of patients must remain on therapy indefinitely, highlighting challenges associated with the retention of a residual pool of BCR-ABL1-positive leukemic stem cells (LSCs) (Mahon et al., The Lancet 2018; Ross et al., Blood 2013). Previously we have shown that, while ABL1 TKIs can effectively inhibit BCR-ABL1 kinase activity and reduce CML disease burden, LSCs avoid ABL1 TKI-induced cell death via alternate signaling pathways such as MEK/ERK and are able to persist (Corbin et al., JCI 2011). Detecting changes in phospho-activation of such alternative pathways at the single cell level using primary CML samples has yet to be characterized and will inform therapeutic options for complete disease eradication. Here, we dissect phospho-signaling diversity with single cell level granularity across heterogeneous subpopulations of CML stem (CD34+CD38-) and progenitor (CD34+CD38+) cells upon drug treatment using a new quantitative imaging method that allows for sensitive detection of critical changes in signaling (Jacob et al., Scientific Reports 2016). Briefly, primary CD34+ cells were isolated from newly diagnosed TKI naïve CML patients by MACS column and cultured ex-vivo in the presence of imatinib and MEK inhibitors (trametinib or cobimetinib), either alone or in combination. Cells were then assessed for apoptosis by flow cytometry annexin assay, followed by fixing/staining for CD38, pCRKL, and pERK, and quantified for changes in signaling upon treatment by quantitative imaging method. This new phospho-imaging method allows for low cell number input and maximizes data output per sample, opening opportunities to assess translational applicability of combinations in a highly biologically relevant context of small but critical LSC populations. Quantification of phospho-signal revealed that pCRKL was reduced in a dose dependent manner across a range of concentrations of imatinib in both CML stem and progenitor cells. In contrast, while pERK levels were also reduced with imatinib treatment, a subset of CML stem cells exhibited high levels of pERK signaling even at high (5uM) concentrations of imatinib. Combination treatment with imatinib and trametinib or cobimetinib markedly increased apoptosis in CML CD34+ cells compared to each single agent. Within the stem cell population, median pERK fluorescence was significantly reduced with imatinib or trametinib compared to untreated control (p<0.0001), and further reduced with the combination compared to each single agent (p<0.0001 and p=0.04, respectively). Furthermore, the percent of cells with pERK levels below the untreated median value was 87%, 90% and 97% upon treatment with imatinib, trametinib or the combination respectively. Together, our data suggest that variable levels of ERK phosphorylation exist within the CML stem and progenitor compartments, including a subset of CML stem cells that exhibit persistently high levels of pERK despite effective suppression of BCR-ABL1 kinase activity by imatinib. This stem cell persistence warrants the need for combination strategies to inhibit BCR-ABL1 and MEK/ERK for their complete elimination.
Disclosures
Tyner: AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Leap Oncology: Equity Ownership; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Agios: Research Funding. Druker:Celgene: Consultancy; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oregon Health & Science University: Patents & Royalties; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Novartis Pharmaceuticals: Research Funding; ARIAD: Research Funding; Millipore: Patents & Royalties; Amgen: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding.