Characterization of Imatinib Resistant CML Leukemic Stem/Initiating Cells and Their Sensitivity to CBP/Catenin Antagonists.

The development of the tyrosine kinase inhibitor Imatinib (IM) represents a milestone in CML (Chronic Myeloid Leukemia) treatment. However, it is not curative and patients develop IM resistance. IM resistance has been previously correlated with the emergence of drug-resistant LIC/LSC (Leukemia Initiating Cell/Leukemia Stem Cell) and increased nuclear catenin levels and enhanced Wnt signaling. It has been demonstrated previously that drug resistant CML LIC/LSC can be safely eliminated both in vitro and in vivo via disruption of the CBP/catenin interaction, utilizing the highly biochemically selective small molecule CBP/catenin antagonist ICG- 001. Here, we utilized an in vitro IM selection of primary CML patients' samples to identify drug-resistant LIC/LSC populations. In this report, we characterized the drug-resistant CML LIC/LSC population using FACS, Smartchip qPCR and colony assays to analyze cell surface markers, transcriptomics and function. As opposed to previous characterization of the CML leukemic stem cell population as being either CD34+CD38- or CD34+CD38+, the in vitro selected Imatinib resistant (IM-R) CML LSC population was consistently CD34-CD38-. In Long-Term Culture Initiating Cell assay (LTC-IC, a surrogate assay for long term repopulating stem cells), our results suggest that the CBP/catenin antagonist ICG- 001 sensitizes LIC/LSC to IM treatment by forced differentiative elimination of the CML LIC/LSC population. In vitro selected IM resistant cells are negative for both CD34 and CD38 by FACS analysis. These cells acquire CD34/CD38 expression after co-culture with stromal cells. CBP/catenin antagonist ICG-001 facilitates IM function in eliminating these cells.

Zhao Y ，Wu K ，Wu Y ，Melendez E ，Smbatyan G ，Massiello D ，Kahn M ... -  《-》

CBP/catenin antagonist safely eliminates drug-resistant leukemia-initiating cells.

Zhao Y ，Masiello D ，McMillian M ，Nguyen C ，Wu Y ，Melendez E ，Smbatyan G ，Kida A ，He Y ，Teo JL ，Kahn M ... -  《-》

Selective and effective targeting of chronic myeloid leukemia stem cells by topoisomerase II inhibitor etoposide in combination with imatinib mesylate in vitro.

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs) have improved chronic myeloid leukemia (CML) patient survival markedly but fail to eradicate quiescent CML leukemia stem cells (LSCs). Thus, strategies targeting LSCs are required to induce long-term remission and achieve cure. Here, we investigated the ability of topoisomerase II (Top II) inhibitor etoposide (Eto) to target CML LSCs. Treatment with Eto combined with IM markedly induced apoptosis in primitive CML CD34+ CD38- stem cells resistant to eradication by IM alone, but not in normal hematopoietic stem cells, CML and normal mature CD34- cells, and other leukemia and lymphoma cell lines. The interaction of IM and Eto significantly inhibited phosphorylation of PDK1, AKT, GSK3, S6, and ERK proteins; increased the expression of pro-apoptotic gene Bax; and decreased the expression of anti-apoptotic gene c-Myc in CML CD34+ cells. Top II inhibitors treatment represents an attractive approach for targeting LSCs in CML patients undergoing TKIs monotherapy.

Liu MY ，Wang WZ ，Liao FF ，Wu QQ ，Lin XH ，Chen YH ，Cheng L ，Jin XB ，Zhu JY ... -  《-》

Dual inhibition of Bcr-Abl and Hsp90 by C086 potently inhibits the proliferation of imatinib-resistant CML cells.

Although tyrosine kinase inhibitors (TKI) such as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of patients with chronic myelogenous leukemia (CML), TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition. Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation, and immunoblotting were applied to examine Hsp90 function. Colony-forming unit, long-term culture-initiating cells (LTC-IC), and flow cytometry were used to test CML progenitor and stem cells. Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, it inhibited the growth of both imatinib-sensitive and -resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34(+)CD38(+) and CD34(+)CD38(-) cells in vitro. Moreover, C086 could decrease the number of CD45(+), CD45(+)CD34(+)CD38(+), and CD45(+)CD34(+)CD38(-) cells in CML NOD-SCID mice. Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs.

Wu L ，Yu J ，Chen R ，Liu Y ，Lou L ，Wu Y ，Huang L ，Fan Y ，Gao P ，Huang M ，Wu Y ，Chen Y ，Xu J ... -  《-》

A Deeply Quiescent Subset of CML LSC depend on FAO yet Avoid Deleterious ROS by Suppressing Mitochondrial Complex I.

Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures. Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset. Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib. We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.

Chimge NO ，Chen MH ，Nguyen C ，Zhao Y ，Wu X ，Gonzalez PG ，Ogana H ，Hurwitz S ，Teo JL ，Chen X ，Du J ，Jin V ，Kim YM ，Ono M ，Argüello RJ ，Kahn M ... -  《-》