ALBERT

Description: Introduction: The BCR-ABL1 fusion gene is a causative oncogene in chronic myeloid leukemia (CML) and 30-50% of acute lymphoblastic leukemia cases. Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations and become resistant to ABL TKI therapy. In particular, the ABL kinase domain mutation T315I is resistant to imatinib and second-generation ABL TKIs (e.g., nilotinib, dasatinib, and bostinib). Accordingly, this mutation is often found in patients with TKI-resistant disease. A third-generation ABL TKI, ponatinib, was recently developed. Ponatinib is a potent oral tyrosine kinase inhibitor that affects both unmutated and mutated BCR-ABL; it is effective against T315I mutant cells and has been approved for TKI-resistant or intolerant CML and Ph-positive ALL patients.Recently, the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib was found to exhibit anti-leukemic activity against T315I-mutant disease. Axitinib is an orally active and potent TKI of VEGFRs 1, 2, and 3 and inhibits BCR-ABL1, especially the T315I variant, via a distinct binding conformation. Materials and methods: In this study, we investigated whether axitinib could suppress ponatinib-resistant compound mutant cells harboring the T315I mutation and primary samples. Results: A 72 h axitinib treatment inhibited the growth of Ba/F3 T315I cells (Figure 1A). Immunoblot analysis of axitinib-treated cells revealed dose-dependent decreases in BCR-ABL, the downstream molecule CrkL, and ribosomal S6 protein phosphorylation and increases in caspase 3 and Poly (ADP-ribose) polymerase (PARP) activity. Ponatinib and axitinib also induced apoptosis, significantly increased caspase activity, and reduced Akt activity. In contrast, clinically available concentrations of axitinib did not inhibit the growth of ponatinib-resistant Ba/F3 cells. Immunoblot analysis revealed that BCR-ABL, Crk-L, and S6 kinase phosphorylation were not inhibited by axitinib or ponatinib. Similarly, no increase in caspase activity or decrease in Akt activity was observed following axitinib treatment, and neither ponatinib nor axitinib affected apoptosis in these cells. We next evaluated primary T315I-mutant and ponatinib-resistant compound mutant samples. Axitinib potently inhibited the growth of T315I mutant primary cells in a dose-dependent manner. Immunoblot analysis further revealed reduced Crk-L and S6 kinase phosphorylation after axitinib or ponatinib treatment. In contrast, the growth of ponatinib-resistant primary cells was not affected by ponatinib or axitinib. Immunoblotting revealed that neither ponatinib nor axitinib affected the phosphorylation of Crk-L and S6 kinase in ponatinib-resistant cells. Conclusion: In CML, ABL TKI resistance is frequently caused by ABL kinase domain mutations. The T315I mutation is resistant to all ABL TKIs except ponatinib. Although axitinib, which is currently being investigated for efficacy in patients with Ph-positive T315I-mutant leukemia, induced apoptosis in T315I-mutant cells, it was ineffective against cells with a compound mutation including T315I. Current evidence to direct the management of ABL TKI-resistant disease, particularly those harboring T315I and compound mutations, is limited. New molecular-targeted drugs and an understanding of ABL TKI resistance mechanisms are required to apply an appropriate therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. In eukaryotic cells, histone acetylation/deacetylation is important in transcriptional regulation. Chromatin acetylation is controlled by the opposing effects of two families of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDACs). Deregulation of HDAC activity may be a cause of malignant disease in humans. Phosphoinositide 3-kinase (PI3K) pathway also regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. CUDC-907 is an oral inhibitor of class I PI3K as well as class I and II HDAC enzymes. CUDC-907 is currently being investigated in a pivotal phase 1 clinical trial against hematological malignancies such as malignant lymphoma. We suggested that CUDC-907 mediated inhibition PI3K and HDAC activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. Materials and methods: In this study, we investigated the combination therapy with a CUDC-907 and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells and primary samples. Results: The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5). We found that mRNA of PI3K subunit is significantly increased after a co-culture with HS-5 in K562 and primary CD34 positive CML samples. 72 h treatment of CUDC-907 exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We examined the intracellular signaling after treatment of CUDC-907. Phosphorylation of JNK, histone acetylation and activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. Anti-apoptotic protein, Mcl-1 was decreased in a dose dependent. We next investigated the efficacy between imatinib and CUDC-907 by using these cell line. Combined treatment of K562 cells with imatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after imatinib and CUDC-907 treatment. We investigated the CUDC-907 activity against T315I positive cells. CUDC-907 potently induced cell growth inhibition of Ba/F3 T315I cells in a dose dependent manner. Combined treatment of Ba/F3 T315I cells with ponatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced after ponatinib and CUDC-907 treatment. To assess the activity of ponatinib and CUDC-907, we performed to test on tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of CUDC-907 inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of CUDC-907 exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells, T315I mutant primary samples and CD34 positive CML samples. Conclusion: These results indicated that administration of the dual PI3K and HDAC inhibitor, CUDC-907 may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures No relevant conflicts of interest to declare.

Description: Background: The inhibition of BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has markedly improved the prognosis of chronic myeloid leukemia (CML). Recently, it has been recognized that some CML patients with a complete molecular response (CMR) are able to maintain treatment-free remission (TFR) after discontinuation of TKIs. However, no predictive prognostic factors for successful discontinuation of the treatment have yet been identified. We set out to further clarify the role of predictive biomarkers in molecular relapse and non-relapse after ABL TKI discontinuation. Materials and methods: Patients in sustained CMR (MR 4.5) undergoing TKI therapy were eligible for inclusion in the study. Molecular relapse was defined as loss of major molecular response (MMR) of at least one point. Genomic DNA was obtained from whole blood using a DNA Extractor WB Kit (Wako, Osaka, Japan), and was subjected to polymerase chain reaction (PCR) amplification using primers designed to detect a deletion site (2903 bp) in intron two of the BCL2L11 gene (forward: 5′-AATACCACAGAGGCCCACAG-3′; reverse: 5′-GCCTGAAGGTGCTGAGAAAG-3′) and JumpStart RedAccuTaq LA DNA polymerase (Sigma Aldrich, St. Louis, MO, USA). Results: 32 CML patients (17 men, 15 women, median age 58.4 years) were included in this study (Sokal category; low 24, intermediate 7, high 1). Six patients were treated with IFNα before TKI treatment, and 3 were treated with IFNα after stopping TKI. Median duration from TKI initiation to discontinuation was 79.3 months (range; 22 to 138 months); median duration of CMR before TKI discontinuation was 47.3 months (range; 5 to 97 months). Seven patients showed loss of MMR; 6 relapsed within 6 months and one showed late relapse at 25 months after discontinuation. The cumulative incidence of MMR loss was estimated as 18.8% at 12 months and at 24 months. Fluctuation of BCR-ABL transcript levels below the MMR threshold (〉 two consecutive positive values) was observed in 6.25% of patients at 24 months after ABL TKI discontinuation. Treatment-free remission was estimated as 81.2% at 12 months and at 24 months. The median period of restoration of second CMR was 6.0 months in re-treated patients. No patient died during the follow-up period. TKI-free remission was estimated as 78.1% at 30 months. There was only a significant difference in BCL2L11 (BIM) deletion polymorphism between the patients who maintained and those who lost MMR (p = 0.0253). No significant difference was observed in prior IFNα therapy, time to complete cytogenetic response (CCyR), time to MMR, and time to CMR between relapsing and non-relapsing patients. Conclusion: Our study shows a specific association between BCL2L11 (BIM) deletion polymorphism and clinical outcome after ABL TKI discontinuation in patients with long-lasting molecular undetectable residual disease. BCL2L11 (BIM) deletion polymorphism may predict relapse after ABL TKI discontinuation, which may have an impact on future ABL TKI discontinuation trials. These results further illustrate the importance of single nucleotide polymorphisms in successful long-term treatment of CML. Disclosures Ohyashiki: Bristol-Myers Squibb KK : Research Funding, Speakers Bureau; Novartis KK: Research Funding, Speakers Bureau.