Antitumor efficacy of arsenic/interferon in preclinical models of chronic myeloid leukemia resistant to tyrosine kinase inhibitors.

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.

The synthetic retinoid ST1926 as a novel therapeutic agent in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Despite multiple attempts at intensifying chemotherapeutic approaches to treatment, only moderate improvements in survival have been made for patients with advanced disease. Retinoic acid is a differentiation agent that has shown some antitumor efficacy in RMS cells in vitro; however, the effects are of low magnitude. E-3-(4'-hydroxyl-3'-adamantylbiphenyl-4-yl) acrylic acid (ST1926) is a novel orally available synthetic atypical retinoid, shown to have more potent activity than retinoic acid in several types of cancer cells. We used in vitro and in vivo models of RMS to explore the efficacy of ST1926 as a possible therapeutic agent in this sarcoma. We found that ST1926 reduced RMS cell viability in all tested alveolar (ARMS) and embryonal (ERMS) RMS cell lines, at readily achievable micromolar concentrations in mice. ST1926 induced an early DNA damage response (DDR), which led to increase in apoptosis, in addition to S-phase cell cycle arrest and a reduction in protein levels of the cell cycle kinase CDK1. Effects were irrespective of TP53 mutational status. Interestingly, in ARMS cells, ST1926 treatment decreased PAX3-FOXO1 fusion oncoprotein levels, and this suppression occurred at a post-transcriptional level. In vivo, ST1926 was effective in inhibiting growth of ARMS and ERMS xenografts, and induced a prominent DDR. We conclude that ST1926 has preclinical efficacy against RMS, and should be further developed in this disease in clinical trials.

ST1926, an orally active synthetic retinoid, induces apoptosis in chronic myeloid leukemia cells and prolongs survival in a murine model.

The tyrosine kinase inhibitor, imatinib, is the first line of treatment for chronic myeloid leukemia (CML) patients. Unfortunately, patients develop resistance and relapse due to bcr-abl point mutations and the persistence of leukemia initiating cells (LIC). Retinoids regulate vital biological processes such as cellular proliferation, apoptosis, and differentiation, in particular of hematopoietic progenitor cells. The clinical usage of natural retinoids is hindered by acquired resistance and undesirable side effects. However, bioavailable and less toxic synthetic retinoids, such as the atypical adamantyl retinoid ST1926, have been developed and tested in cancer clinical trials. We investigated the preclinical efficacy of the synthetic retinoid ST1926 using human CML cell lines and the murine bone marrow transduction/transplantation CML model. In vitro, ST1926 induced irreversible growth inhibition, cell cycle arrest and apoptosis through the dissipation of the mitochondrial membrane potential and caspase activation. Furthermore, ST1926 induced DNA damage and downregulated BCR-ABL. Most importantly, oral treatment with ST1926 significantly prolonged the longevity of primary CML mice, and reduced tumor burden. However, ST1926 did not eradicate LIC, evident by the ability of splenocytes isolated from treated primary mice to develop CML in untreated secondary recipients. These results support a potential therapeutic use of ST1926 in CML targeted therapy.

Effective targeting of chronic myeloid leukemia initiating activity with the combination of arsenic trioxide and interferon alpha.

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.

EAPB0503, a novel imidazoquinoxaline derivative, inhibits growth and induces apoptosis in chronic myeloid leukemia cells.

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.

An Integrative Analysis of microRNA and mRNA Profiling in CML Stem Cells.

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.