Targeting SYK kinase‐dependent anti‐apoptotic resistance pathway in B‐lineage acute lymphoblastic leukaemia (ALL) cells with a potent SYK inhibitory pentapeptide mimic

The present study found that the pentapeptide mimic C‐61, targeting the substrate binding P‐site of SYK tyrosine kinase acted as a potent inducer of apoptosis in chemotherapy‐resistant SYK‐expressing primary leukemic B‐cell precursors taken directly from relapsed B‐precursor leukaemia (BPL) patients (but not SYK‐deficient infant pro‐B leukaemia cells), exhibited favourable pharmacokinetics in mice and non‐human primates, and eradicated in vivo clonogenic leukaemia cells in severe combined immunodeficient mouse xenograft models of chemotherapy‐resistant human BPL at dose levels non‐toxic to mice and non‐human primates. These in vitro and in vivo findings provide proof of principle for effective treatment of chemotherapy‐resistant BPL by targeting SYK‐dependent anti‐apoptotic blast cell survival machinery with a SYK P‐Site inhibitor. Further development of C‐61 may provide the foundation for therapeutic innovation against chemotherapy‐resistant BPL.     

Bioenergetic modulation overcomes glucocorticoid resistance in T‐lineage acute lymphoblastic leukaemia

Drug‐resistant forms of acute lymphoblastic leukaemia (ALL) are a leading cause of death from disease in children. Up to 25% of patients with T‐cell ALL (T‐ALL) develop resistance to chemotherapeutic agents, particularly to glucocorticoids (GCs), a class of drug to which resistance is one of the strongest indicators of poor clinical outcome. Despite their clinical importance, the molecular mechanisms that underpin GC resistance and leukaemia relapse are not well understood. Recently, we demonstrated that GC‐resistance is associated with a proliferative metabolism involving the up‐regulation of glycolysis, oxidative phosphorylation and cholesterol biosynthesis. Here we confirm that resistance is directly associated with a glycolytic phenotype and show that GC‐resistant T‐ALL cells are able to shift between glucose bioenergetic pathways. We evaluated the potential for targeting these pathways in vitro using a glycolysis inhibitor, 2‐deoxyglucose (2DG), and the oxidative phosphorylation inhibitor oligomycin in combination with methylprednisolone (MPRED). We found that oligomycin synergized with MPRED to sensitize cells otherwise resistant to GCs. Similarly we observed synergy between MPRED and simvastatin, an inhibitor of cholesterol metabolism. Collectively, our findings suggest that dual targeting of bioenergetic pathways in combination with GCs may offer a promising therapeutic strategy to overcome drug resistance in ALL.     

Bis(1H-indol-2-yl)methanones are effective inhibitors of FLT3-ITD tyrosine kinase and partially overcome resistance to PKC412A in vitro

Inhibition of the mutated fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase is a promising therapeutic strategy in acute myeloid leukaemia (AML). However, development of resistance to FLT3 tyrosine kinase inhibitors (TKI), such as PKC412A, has been described recently. This observation may have an increasing impact on the duration of response and relapse rates in upcoming clinical trials employing FLT3-TKI. Herein we investigated two representatives of a novel class of FLT3-TKI: Bis(1 H -indol-2-yl)methanones. Both compounds effectively induced apoptosis in FLT3-internal tandem duplicate (ITD)-transfected murine myeloid cells and in primary FLT3-ITD positive blasts. Combination of both compounds with chemotherapy revealed synergistic effects in apoptosis assays. The compounds did not show significant toxicity in human bone marrow cells derived from healthy donors. Compound102 overcame resistance to PKC412 within a non-myelotoxic dose-range. Western Blotting experiments of 32D-FLT3-ITD cells showed dose-dependent dephosphorylation of FLT3-ITD and of its downstream targets STAT5, AKT and ERK upon incubation with either compound. In conclusion, bis(1 H -indol-2-yl)methanones overcome resistance mediated by FLT3-ITD mutations at position N676 and show strong efficacy in FLT3-ITD-positive cells alone as well as in combination with chemotherapy. We propose that further development of methanone compounds overcoming resistance to currently established FLT3-TKIs is an important step forward to an anticipated need within our future therapeutic algorithm in FLT3-ITD-positive AML.     

HMGA2 as a potential molecular target in KMT2A‐AFF1‐positive infant acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) in infants is an intractable cancer in childhood. Although recent intensive chemotherapy progress has considerably improved ALL treatment outcome, disease cure is often accompanied by undesirable long‐term side effects, and efficient, less toxic molecular targeting therapies have been anticipated. In infant ALL cells with KMT2A (MLL) fusion, the microRNA let‐7b (MIRLET7B) is significantly downregulated by DNA hypermethylation of its promoter region. We show here that the expression of HMGA2, one of the oncogenes repressed by MIRLET7B, is reversely upregulated in infant ALL leukaemic cells, particularly in KMT2A‐AFF1 (MLL‐AF4) positive ALL. In addition to the suppression of MIRLET7B, KMT2A fusion proteins positively regulate the expression of HMGA2. HMGA2 is one of the negative regulators of CDKN2A gene, which encodes the cyclin‐dependent kinase inhibitor p16^INK4A. The HMGA2 inhibitor netropsin, when combined with demethylating agent 5‐azacytidine, upregulated and sustained the expression of CDKN2A, which resulted in growth suppression of KMT2A‐AFF1‐expressing cell lines. This effect was more apparent compared to treatment with 5‐azacytidine alone. These results indicate that the MIRLET7B‐HMGA2‐CDKN2A axis plays an important role in cell proliferation of leukaemic cells and could be a possible molecular target for the therapy of infant ALL with KMT2A‐AFF1.     

Simultaneous targeting of MCL1 and ABCB1 as a novel strategy to overcome drug resistance in human leukaemia

Drug resistance is a major obstacle to chemotherapy success in leukaemia. Although ABCB1 ( MDR1 ) overexpression represents a critical mechanism of drug resistance, modulation of ABCB1 shows unsatisfactory clinical outcome. Recent studies showed that MCL1 was upregulated in numerous haematological and solid tumour malignancies. The present study found that patients with newly diagnosed or relapsed/refractory leukaemia expressed higher MCL1 levels than patients that were in complete remission. We demonstrated that overexpression of MCL1 decreased sensitivity of human leukaemia cell lines to cytotoxic drugs and inhibited drug-induced apoptosis. Specific downregulation of MCL1 via RNA interference sensitized multidrug resistant leukaemia cells towards chemotherapy and induced apoptosis. Our study also demonstrated that MCL1 and ABCB1 mediated drug resistance through different mechanisms and the depletion of both MCL1 and ABCB1 showed an additive effect in reversing drug resistance and promoting drug-induced apoptosis. Thus, this study documented an important role of MCL1 in drug resistance and apoptosis. Simultaneous targeting of MCL1 and ABCB1 could be a novel approach to overcome drug resistance in leukaemia.     

Poor responses to tyrosine kinase inhibitors in a child with precursor B‐cell acute lymphoblastic leukemia with SNX2‐ABL1 chimeric transcript

In addition to BCR, various rare fusion partners for the ABL1 gene have been reported in leukemia. We have identified the fusion gene SNX2‐ABL1 in a pediatric case of acute lymphoblastic leukemia (ALL), which has only once previously been reported in an adult patient. Cytogenetic analysis detected this fusion gene arising from a t(5;9)(q22;q34) translocation. ALL cells carrying a SNX2‐ABL1 fusion exhibited a BCR‐ABL1+ ALL‐like gene expression profile. The patient poorly responded to dasatinib but partially responded to imatinib. Treatment using tyrosine kinase inhibitors requires further investigation to optimize the genotype‐based treatment stratification for patients with SNX2‐ABL1 fusion.     

Rapid identification of BCR/ABL1‐like acute lymphoblastic leukaemia patients using a predictive statistical model based on quantitative real time‐polymerase chain reaction: clinical,prognostic and therapeutic implications

BCR/ABL1‐like acute lymphoblastic leukaemia (ALL) is a subgroup of B‐lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1‐like ALL cases and used their expression values – assessed by quantitative real time‐polymerase chain reaction (Q‐RT‐PCR) in 26 BCR/ABL1‐like and 26 non‐BCR/ABL1‐like cases to build a statistical “BCR/ABL1‐like predictor”, for the identification of BCR/ABL1‐like cases. By screening 142 B‐lineage ALL patients with the “BCR/ABL1‐like predictor”, we identified 28/142 BCR/ABL1‐like patients (19·7%). Overall, BCR/ABL1‐like cases were enriched in JAK/STAT mutations (P < 0·001), IKZF1 deletions (P < 0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P = 0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1‐like cases identified by the BCR/ABL1‐like predictor achieved a lower rate of complete remission (P = 0·014) and a worse event‐free survival (P = 0·0009) compared to non‐BCR/ABL1‐like ALL. Consistently, primary cells from BCR/ABL1‐like cases responded in vitro to ponatinib. We propose a simple tool based on Q‐RT‐PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1‐like ALL cases at diagnosis.     

Acute myeloid/T‐lymphoblastic leukaemia (AMTL): a distinct category of acute leukaemias with common pathogenesis in need of improved therapy

Advances in the classification of acute leukaemias have led to improved outcomes for a substantial fraction of patients. However, chemotherapy resistance remains a major problem for specific subsets of acute leukaemias. Here, we propose that a molecularly distinct subtype of acute leukaemia with shared myeloid and T cell lymphoblastic features, which we term acute myeloid/T‐lymphoblastic leukaemia (AMTL), is divided across 3 diagnostic categories owing to variable expression of markers deemed to be defining of myeloid and T‐lymphoid lineages, such as myeloperoxidase and CD3. This proposed diagnostic group is supported by (i) retained myeloid differentiation potential during early T cell lymphoid development, (ii) recognition that some cases of acute myeloid leukaemia (AML) harbour hallmarks of T cell development, such as T‐cell receptor gene rearrangements and (iii) common gene mutations in subsets of AML and T cell acute lymphoblastic leukaemia (T‐ALL), including WT1, PHF6, RUNX1 and BCL11B. This proposed diagnostic entity overlaps with early T cell precursor (ETP) T‐ALL and T cell/myeloid mixed phenotype acute leukaemias (MPALs), and also includes a subset of leukaemias currently classified as AML with features of T‐lymphoblastic development. The proposed classification of AMTL as a distinct entity would enable more precise prospective diagnosis and permit the development of improved therapies for patients whose treatment is inadequate with current approaches.     

Melatonin sensitizes human cervical cancer HeLa cells to cisplatin‐induced cytotoxicity and apoptosis: effects on oxidative stress and DNA fragmentation

Melatonin has antitumor activity via several mechanisms including its antiproliferative and pro‐apoptotic effects as well as its potent antioxidant actions, although recent evidence has indicated that melatonin may perform pro‐oxidant actions in tumor cells. Therefore, melatonin may be useful in the treatment of tumors in association with chemotherapy drugs. This study was intended to evaluate the in vitro effect of melatonin on the cytotoxic and pro‐apoptotic actions of various chemotherapeutic agents in cervical cancer HeLa cells. Herein, we found that both melatonin and three of the chemotherapeutic drugs tested, namely cisplatin (CIS), 5‐fluorouracil (5‐FU), and doxorubicin, induced a decrease in HeLa cell viability. Furthermore, melatonin significantly increased the cytotoxic effect of such chemotherapeutic agents. Consistently, costimulation of HeLa cells with any chemotherapeutic agent in the presence of melatonin further increased caspase‐3 activation, particularly in CIS‐ and 5‐FU‐challenged cells. Likewise, concomitant treatments with melatonin and CIS significantly enhanced the ratio of cells entering mitochondrial apoptosis due to reactive oxygen species (ROS) overproduction, substantially augmented the population of apoptotic cells, and markedly enlarged DNA fragmentation compared to the treatments with CIS alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in 5‐FU‐stimulated HeLa cells, as suggested by slight increments in the percentage of cells stimulated for ROS production and in the proportion of early apoptotic cells compared to the treatments with 5‐FU alone. In summary, our findings provided evidence that in vitro melatonin strongly enhances CIS‐induced cytotoxicity and apoptosis in HeLa cells and, hence, the indoleamine could be potentially applied to cervical cancer treatment as a powerful synergistic agent.     

Prognostic relevance of dic(9;20)(p11;q13) in childhood B‐cell precursor acute lymphoblastic leukaemia treated with Berlin‐Frankfurt‐Münster (BFM) protocols containing an intensive induction and post‐induction consolidation therapy

The presence of a dicentric chromosome dic(9;20) has been reported to have an unfavourable prognosis in children with B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL). As outcome may be influenced by type and composition of treatment, we analyzed 19 BCP‐ALL patients with dic(9;20) who have been treated with ALL‐BFM (Berlin‐Frankfurt‐Münster) protocols that included a 4‐drug induction and subsequent consolidation therapy. All patients were good responders to prednisone and in complete remission after induction therapy. Eight patients had no molecular disease after induction and another eight patients had levels ≤10^?4 after consolidation therapy. After a median follow‐up of 3·4 years, probabilities of 5‐year event‐free and overall survival were 75 ± 11% and 94 ± 6%, respectively. Of note, there was a tendency for extramedullary disease in case of relapse (two of three relapses with central nervous system involvement). In conclusion, in the context of ALL‐BFM protocols dic(9;20)‐positivity appeared to have a favourable prognosis, which could be due to a dose‐ and time‐intensified induction and induction consolidation therapy. Given that in vitro studies have shown high cellular sensitivity of dic(9;20)‐positive leukemic blasts to l ‐asparaginase and cytarabine, it is reasonable to speculate that both drugs, as given early during BFM‐like induction and consolidation therapy, may have contributed to this good outcome.     

The c-Jun-N-terminal-Kinase inhibitor SP600125 enhances the butyrate derivative D1-induced apoptosis via caspase 8 activation in Kasumi 1 t(8;21) acute myeloid leukaemia cells

We recently showed that the histone deacetylase inhibitor D1 induced apoptosis in the t(8;21) Kasumi 1 acute myeloid leukaemia (AML) cell line and activated caspase 9. The present study characterised the effects of the combined administration of D1 with PD98059, SB203580 or SP600125, specific inhibitors of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 or Jun N-terminal kinase (JNK), respectively. Among these inhibitors, SP600125 was the only one to markedly induce apoptosis and decrease cell proliferation. These experiments showed that SP600125 activated caspase 8 and confirmed that D1 activated the intrinsic pathway of apoptosis, as caspase 8 was not affected while Bcl-2 was down-regulated following D1 administration. The combination of the two drugs enhanced caspase-8 activation and induced apoptosis in an additive fashion. JNK was constitutively activated in the Kasumi 1, NB4, HL60 and THP-1 human AML cell lines, as well as in primary blasts from a t(8;21) AML patient. In all these cells, the pro-apoptotic effect of the two drugs alone was increased when they were combined. On this basis, the combined administration of D1 with SP600125 seems to be very promising as a potential anti-leukaemic tool in AML.