Imatinib is the standard of care in chronic meloid leukemia (CML) therapy. However, imatinib is not curative since most patients who discontinue therapy relapse indicating that leukemia initiating cells (LIC) are resistant. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. Arsenic trioxide was suggested to decrease CML LIC. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction=transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Finally, in vivo, combined interferon and arsenic treatment, but not single agents, prolonged the survival of primary CML mice. Importantly, the combination severely impaired engraftment into untreated secondary recipients, with some recipients never developing the disease, demonstrating a dramatic decrease in CML LIC activity. Arsenic=IFN effect on CML LIC activity was significantly superior to that of imatinib. These results support further exploration of this combination, alone or with imatinib aiming at achieving CML eradication rather than long-term disease control.
Imatinib, the first-generation tyrosine kinase inhibitor, revolutionized the therapeutic management of chronic myeloid leukemia (CML) and is highly effective in inducing remissions and prolonging the survival of CML patients. However, one-third of patients develop intolerance or resistance to treatment, and CML stem cells remain insensitive to this therapy, leading almost inevitably to relapse upon treatment discontinuation. Imidazoquinoxalines are imiquimod derivatives that induce growth inhibition and induction of caspase-dependent apoptosis in melanoma and T-cell lymphoma cells. We investigated the effects of EAPB0203 and EAPB0503, two novel imidazoquinoxaline derivatives, on human CML cell lines and showed that they induced a dose-dependent and time-dependent cell growth inhibition. EAPB0503 proved more potent and induced a specific cell cycle arrest in mitosis in CML cells and direct activation of apoptosis as evidenced by increased pre-G0 population, breakdown of mitochondrial membrane potential, PARP cleavage, and DNA breakage. Interestingly, EAPB0503 decreased BCR-ABL oncoprotein levels. The combination of EAPB0503 with imatinib synergized to inhibit the proliferation of CML cells, and most importantly, EABP0503 inhibited the proliferation of imatinib-resistant CML cells, offering promising therapeutic modalities that would circumvent resistance to tyrosine kinase inhibitors and improve the prognosis of CML.
Imatinib is the standard of care in the treatment of Chronic Myeloid Leukemia (CML) patients. However, imatinib is not curative since most patients who discontinue therapy will relapse, stressing the need for novel therapies that target leukemia initiating cells (LIC). Arsenic trioxide induces complete remission in patients with acute promyelocytic leukemia (APL). We previously demonstrated that, through full degradation of PML-RARA, the combination of arsenic and retinoic acid eradicates APL LICs and cures the disease. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Moreover, in a murine transplantation model of CML, we show that arsenic/IFN prolonged survival of primary leukemic mice and sharply diminished transplantation of CML cells in secondary recipients, pointing to exhaustion of CML LICs. Our results also suggest that IFN/arsenic may clear LICs independently from oncoprotein catabolism. Interestingly, arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib.These studies plea for a clinical exploration of this combination, knowing that IFN and arsenic have both shown clinical activity in CML, alone or in combination with imatinib. Disclosures: No relevant conflicts of interest to declare.
Background Tyrosine kinase inhibitors (TKIs) are the standard treatment for chronic myeloid leukemia (CML). Despite their clinical success, TKIs are faced with challenges such as treatment resistance, which may be driven by kinase domain mutations, and frequent disease relapse upon the cessation of treatment. The combination of arsenic trioxide (ATO) and interferon‐α (IFN) was previously demonstrated to inhibit proliferation and induce apoptosis in CML cell lines, prolong the survival of primary wild‐type CML mice, and dramatically decrease the activity of leukemia‐initiating cells (LICs). Methods The ATO/IFN combination was tested in vitro on imatinib (IMN)‐resistant K562‐R and Ar230‐R cells. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling assays were used to evaluate proliferation and apoptosis, respectively. The acridine orange assay was used to assess autophagy, and quantitative reverse transcription–polymerase chain reaction was used to assess the involvement of the hedgehog (Hh) pathway. In vivo, a retroviral transduction/transplantation T315I BCR‐ABL CML mouse model was used to assay the effect of the treatment on survival, tumor burden (histopathology and blood counts), and LIC activity (secondary transplantation). Results In vitro, ATO/IFN synergized to inhibit proliferation and induce apoptosis of IMN‐resistant cells with variant modes of resistance. Furthermore, the preclinical effects of ATO/IFN were associated with induction of autophagy along with inhibition of the Hh pathway. Most remarkably, ATO/IFN significantly prolonged the survival of primary T315I‐CML mice and displayed a dramatic impairment of disease engraftment in secondary mice, which reflected decreased LIC activity. Conclusions Collectively, the ATO/IFN strategy has been demonstrated to have the potential to lead to durable remissions in TKI‐resistant CML preclinical models and to overcome various TKI‐specific mechanisms of resistance.
Integrative analysis of microRNA (miRNA) and messenger RNA (mRNA) in Chronic Myeloid leukemia (CML) stem cells is an important technique to study the involvement of miRNA and their targets in CML stem cells self-renewal, maintenance, and therapeutic resistance. Here, we describe a simplified integrative analysis using Ingenuity Pathway Analysis software after performing proper RNA extraction, miRNA and mRNA microarray and data analysis.
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