Yosuke Minami

Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial

BACKGROUNDnFirst-line imatinib treatment can be successfully discontinued in patients with chronic myeloid leukaemia after deep molecular response has been sustained for at least 2 years. We investigated the safety and efficacy of discontinuing second-line or subsequent dasatinib after at least 1 year of deep molecular response.nnnMETHODSnThe Dasatinib Discontinuation trial was a prospective multicentre trial done in Japan. Eligible patients taking dasatinib and with confirmed stable deep molecular response were enrolled between April 1, 2011, and March 31, 2012. All patients received dasatinib consolidation therapy for at least 1 year. In those with sustained deep molecular response, dasatinib was discontinued. Patients were followed up every month in year 1 (clinical cutoff), every 3 months in year 2, and every 6 months in year 3 for deep molecular response and immunological profiles. The primary endpoint was the proportion of patients with treatment-free remission at 6 months after discontinuation. Molecular relapse was defined as loss of deep molecular response at any assessment. This study is registered, number UMIN000005130.nnnFINDINGSn88 patients were enrolled in the consolidation phase, 24 were excluded from the discontinuation phase due to fluctuations in BCR-ABL1 transcript levels. One patient was excluded because of positive expression of major and minor BCR-ABL1 transcripts in chronic myeloid leukaemia cells and the detection of minor BCR-ABL1 transcripts during consolidation. Thus, 63 patients discontinued dasatinib treatment. The 25 patients who were excluded from discontinuation continued to receive dasatinib and none showed disease progression. Median follow-up was 20.0 months (IQR 16.5-24.0). Of the 63 patients who discontinued and were not excluded, 30 patients maintained deep molecular response while 33 patients had molecular relapses, all within the first 7 months after discontinuation. The estimated overall treatment-free remission was 49% (95% CI 36-61) at 6 months. No severe treatment-related toxic effects were seen. Treatment was restarted in the 33 patients with relapse; rapid molecular responses were seen in all 33 patients, of whom 29 (88%) regained deep molecular response within 3 months, as did the remaining four by 6 months.nnnINTERPRETATIONnDasatinib discontinuation after sustained deep molecular response for more than 1 year is feasible.nnnFUNDINGnEpidemiological and Clinical Research Information Network (ECRIN).

Combination of Ponatinib with Hedgehog Antagonist Vismodegib for Therapy-Resistant BCR-ABL1–Positive Leukemia

Purpose: The Hedgehog signaling pathway is a key regulator of cell growth and differentiation during development. Whereas the Hedgehog pathway is inactive in most normal adult tissues, Hedgehog pathway reactivation has been implicated in the pathogenesis of several neoplasms including BCR-ABL1–positive leukemia. The clear link between the Hedgehog pathway and BCR-ABL1–positive leukemia led to an effort to identify small molecules to block the pathway. Experimental Design: We investigated the combined effects of vismodegib and ponatinib, a pan-ABL1 kinase inhibitor, in nonobese diabetic/severe-combined immunodeficiency (NOD/SCID) repopulating T315I BCR-ABL1–positive cells in vitro and in vivo. Results: We observed that combination with vismodegib and ponatinib helps to eliminate therapy-resistant NOD/SCID repopulating T315I BCR-ABL1–positive cells. The percentage of CD19-positive leukemia cells in peripheral blood was significantly lower in vismodegib + ponatinib–treated mice than that of the vehicle or ponatinib alone (P < 0.001). Spleen weights were also lower in vismodegib + ponatinib–treated mice than in ponatinib alone (P < 0.05). Overall tumor burden, as assessed by BCR-ABL mRNA from bone marrow cells, was significantly lower in vismodegib + ponatinib–treated mice than in ponatinib alone (P < 0.005). We also found that vismodegib significantly reduced BCR-ABL1–positive leukemia cell self-renewal in vitro as well as during serial transplantation in vivo. Conclusions: The combination with a Smo inhibitor and ABL1 tyrosine kinase inhibitors may help eliminate therapy-resistant T315I BCR-ABL1–positive leukemia cells. Our preclinical results indicate that vismodegib has potential as an important option for controlling minimal residual cells in BCR-ABL1–positive leukemia. Clin Cancer Res; 19(6); 1422–32. ©2012 AACR.

Anti-cancer fatty-acid derivative induces autophagic cell death through modulation of PKM isoform expression profile mediated by bcr-abl in chronic myeloid leukemia.

The fusion gene bcr-abl develops chronic myeloid leukemia (CML), and stimulates PI3K/Akt/mTOR signaling, leading to impaired autophagy. PI3K/Akt/mTOR signaling also plays an important role in cell metabolism. The Warburg effect is a well-recognized hallmark of cancer energy metabolism, and is regulated by the mTOR/c-Myc/hnRNP/PKM signaling cascade. To develop a new strategy for the treatment of CML, we investigated the associations among bcr-abl, the cascade related to cancer energy metabolism, and autophagy induced by a fatty-acid derivative that we had previously reported as being an autophagy inducer. Here we report that a fatty-acid derivative, AIC-47, induced transcriptional repression of the bcr-abl gene and modulated the expression profile of PKM isoforms, resulting in autophagic cell death. We show that c-Myc functioned as a transcriptional activator of bcr-abl, and regulated the hnRNP/PKM cascade. AIC-47, acting through the PPARγ/β-catenin pathway, induced down-regulation of c-Myc, leading to the disruption of the bcr-abl/mTOR/hnRNP signaling pathway, and switching of the expression of PKM2 to PKM1. This switching caused autophagic cell death through an increase in the ROS level. Our findings suggest that AIC-47 induced autophagic cell death through the PPARγ/β-catenin/bcr-abl/mTOR/hnRNP/PKM cascade.

Association between severe toxicity of nilotinib and UGT1A1 polymorphisms in Japanese patients with chronic myelogenous leukemia.

BackgroundNilotinib is a BCR-ABL kinase inhibitor approved for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (CML). The UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphism UGT1A1*28 (*28)/*28 has been linked to an increased risk of hyperbilirubinemia in patients with CML who receive nilotinib. Beside *28, UGT1A1*6 (*6) is another important variant allele in Japanese patients because it is associated with adverse events of irinotecan, metabolized by UGT1A1. We retrospectively investigated the association between severe toxicity of nilotinib and UGT1A1 polymorphisms (*6 and*28) in Japanese patients with CML.Patients and methodsEight patients with cytogenetically confirmed CML who were receiving nilotinib were studied to explore the association of UGT1A1 polymorphisms with severe nilotinib-related toxicity. Genotyping analyses were determined for *6 and *28.ResultsAll 3 patients with the *6/*6 or *6/*28 genotype had severe toxicity, including QT interval prolongation (grade 3), elevated lipase levels (grade 3) plus hyperbilirubinemia (grade 2), and anemia (grade 3) plus hepatic cyst hemorrhage (grade 2) in 1 patient each. Among the 5 patients with the *6/*1 or *1/*1 genotype, 1 had elevated lipase levels (grade 3) and another had severe pain in the lower extremities (grade 3).ConclusionThese findings suggest that UGT1A1 polymorphisms are important determinants of severe toxicity of nilotinib in Japanese patients.

Heme‐related molecules induce rapid production of neutrophil extracellular traps

Pulmonary endothelial cell damages caused by neutrophil overactivation could result in acute lung injuries including transfusion‐related acute lung injury (TRALI). We previously reported that heme‐related molecules derived from hemolysis induced the production of reactive oxygen species from neutrophils. Recently, neutrophil extracellular traps (NETs) have been demonstrated to associate with the onset of TRALI.

Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines.

Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

Small-molecule Hedgehog inhibitor attenuates the leukemia-initiation potential of acute myeloid leukemia cells.

Aberrant activation of the Hedgehog signaling pathway has been implicated in the maintenance of leukemia stem cell populations in several model systems. PF‐04449913 (PF‐913) is a selective, small‐molecule inhibitor of Smoothened, a membrane protein that regulates the Hedgehog pathway. However, details of the proof‐of‐concept and mechanism of action of PF‐913 following administration to patients with acute myeloid leukemia (AML) are unclear. This study examined the role of the Hedgehog signaling pathway in AML cells, and evaluated the in vitro and in vivo effects of the Smoothened inhibitor PF‐913. In primary AML cells, activation of the Hedgehog signaling pathway was more pronounced in CD34+ cells than CD34− cells. In vitro treatment with PF‐913 induced a decrease in the quiescent cell population accompanied by minimal cell death. In vivo treatment with PF‐913 attenuated the leukemia‐initiation potential of AML cells in a serial transplantation mouse model, while limiting reduction of tumor burden in a primary xenotransplant system. Comprehensive gene set enrichment analysis revealed that PF‐913 modulated self‐renewal signatures and cell cycle progression. Furthermore, PF‐913 sensitized AML cells to cytosine arabinoside, and abrogated resistance to cytosine arabinoside in AML cells cocultured with HS‐5 stromal cells. These findings imply that pharmacologic inhibition of Hedgehog signaling attenuates the leukemia‐initiation potential, and also enhanced AML therapy by sensitizing dormant leukemia stem cells to chemotherapy and overcoming resistance in the bone marrow microenvironment.

Perturbation of energy metabolism by fatty-acid derivative AIC-47 and imatinib in BCR-ABL-harboring leukemic cells

In Ph-positive leukemia, imatinib brought marked clinical improvement; however, further improvement is needed to prevent relapse. Cancer cells efficiently use limited energy sources, and drugs targeting cellular metabolism improve the efficacy of therapy. In this study, we characterized the effects of novel anti-cancer fatty-acid derivative AIC-47 and imatinib, focusing on cancer-specific energy metabolism in chronic myeloid leukemia cells. AIC-47 and imatinib in combination exhibited a significant synergic cytotoxicity. Imatinib inhibited only the phosphorylation of BCR-ABL; whereas AIC-47 suppressed the expression of the protein itself. Both AIC-47 and imatinib modulated the expression of pyruvate kinase M (PKM) isoforms from PKM2 to PKM1 through the down-regulation of polypyrimidine tract-binding protein 1 (PTBP1). PTBP1 functions as alternative splicing repressor of PKM1, resulting in expression of PKM2, which is an inactive form of pyruvate kinase for the last step of glycolysis. Although inactivation of BCR-ABL by imatinib strongly suppressed glycolysis, compensatory fatty-acid oxidation (FAO) activation supported glucose-independent cell survival by up-regulating CPT1C, the rate-limiting FAO enzyme. In contrast, AIC-47 inhibited the expression of CPT1C and directly fatty-acid metabolism. These findings were also observed in the CD34(+) fraction of Ph-positive acute lymphoblastic leukemia cells. These results suggest that AIC-47 in combination with imatinib strengthened the attack on cancer energy metabolism, in terms of both glycolysis and compensatory activation of FAO.

Phase I study of glasdegib (PF‐04449913), an oral smoothened inhibitor, in Japanese patients with select hematologic malignancies

The hedgehog signaling pathway regulates multiple morphogenetic processes during embryogenesis. Aberrant activation of the hedgehog pathway signal transduction in adult tissues is associated with the pathogenesis of hematologic malignancies and solid tumors. We report findings from an open‐label, multicenter phase I trial of the selective, small‐molecule hedgehog signaling inhibitor glasdegib (PF‐04449913) in Japanese patients with select advanced hematologic malignancies. Glasdegib was administered as once‐daily oral doses (25, 50 and 100 mg) in 28‐day cycles after a lead‐in dose on Day −5. The primary objectives were to determine first‐cycle dose‐limiting toxicities, safety, vital signs and laboratory test abnormalities. Secondary objectives included evaluation of pharmacokinetics, pharmacodynamics and preliminary evidence of clinical activity of glasdegib. No dose‐limiting toxicities were noted in the 13 patients in the present study. All patients experienced at least one treatment‐emergent, all‐causality adverse event. The most frequent treatment‐related adverse events (observed in ≥3 patients) were dysgeusia (n = 9), muscle spasms (n = 5), alopecia, decreased appetite (n = 4 each), and increased blood creatinine phosphokinase, constipation and diarrhea (n = 3 each). Two deaths occurred during the study and were deemed not to be treatment‐related due to disease progression. Glasdegib demonstrated dose‐proportional pharmacokinetics, marked downregulation of the glioma‐associated transcriptional regulator GLI1 expression in normal skin, and evidence of preliminary clinical activity, although data are limited. Glasdegib was safe and well tolerated across the dose levels tested. It is confirmed that the 100‐mg dose is safe and tolerable in Japanese patients, and this dose level will be examined in the future clinical trial.

A prospective study of the antiemetic effect of palonosetron in malignant lymphoma patients treated with the CHOP regimen

To identify strategies for reducing emesis induced by the CHOP regimen, which includes high-dose steroids, we prospectively evaluated the efficacy of palonosetron in Japanese patients. Palonosetron was administered at a dose of 0.75xa0mg via intravenous injection over 30xa0min before chemotherapy on day 1. Patients kept diaries of chemotherapy-induced nausea and vomiting (CINV) incidence from the start of chemotherapy until 168xa0h afterwards, in which they documented the occurrence and severity of nausea, vomiting, anorexia, and the use of rescue medication. The primary endpoint was the overall occurrence rate of nausea, vomiting, and anorexia; these rates were 56, 12, and 62xa0%, respectively, including all grades. The rates and severity of symptoms tended to worsen 120–168xa0h after completing oral prednisolone. We defined complete response (CR) as no vomiting and no use of rescue therapy. The CR rates of post palonosetron 0.75xa0mg treatment in the acute (0–24xa0h), delayed (24–168xa0h), and overall phases (0–168xa0h) were 86, 66, and 62xa0%, respectively. Antiemetic strategies of CHOP regimen for day 6 and, thereafter, should be investigated.