Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants.

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211-8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients.

Gene expression profiling-based dissection of MLL translocated and MLL germline acute lymphoblastic leukemia in infants.

Acute lymphoblastic leukemia (ALL) in infants (< 1 year) is characterized by a poor prognosis and a high incidence of MLL translocations. Several studies demonstrated the unique gene expression profile associated with MLL-rearranged ALL, but generally small cohorts were analyzed as uniform patient groups regardless of the type of MLL translocation, whereas the analysis of translocation-negative infant ALL remained unacknowledged. Here we generated and analyzed primary infant ALL expression profiles (n = 73) typified by translocations t(4;11), t(11;19), and t(9;11), or the absence of MLL translocations. Our data show that MLL germline infant ALL specifies a gene expression pattern that is different from both MLL-rearranged infant ALL and pediatric precursor B-ALL. Moreover, we demonstrate that, apart from a fundamental signature shared by all MLL-rearranged infant ALL samples, each type of MLL translocation is associated with a translocation-specific gene expression signature. Finally, we show the existence of 2 distinct subgroups among t(4;11)-positive infant ALL cases characterized by the absence or presence of HOXA expression, and that patients lacking HOXA expression are at extreme high risk of disease relapse. These gene expression profiles should provide important novel insights in the complex biology of MLL-rearranged infant ALL and boost our progress in finding novel therapeutic solutions.

Association of high-level MCL-1 expression with in vitro and in vivo prednisone resistance in MLL-rearranged infant acute lymphoblastic leukemia.

MLL-rearranged acute lymphoblastic leukemia (ALL) represents an unfavorable type of leukemia that often is highly resistant to glucocorticoids such as prednisone and dexamethasone. Because response to prednisone largely determines clinical outcome of pediatric patients with ALL, overcoming resistance to this drug may be an important step toward improving prognosis. Here, we show how gene expression profiling identifies high-level MCL-1 expression to be associated with prednisolone resistance in MLL-rearranged infant ALL, as well as in more favorable types of childhood ALL. To validate this observation, we determined MCL-1 expression with quantitative reverse transcription-polymerase chain reaction in a cohort of MLL-rearranged infant ALL and pediatric noninfant ALL samples and confirmed that high-level MCL-1 expression is associated with prednisolone resistance in vitro. In addition, MCL-1 expression appeared to be significantly higher in MLL-rearranged infant patients who showed a poor response to prednisone in vivo compared with prednisone good responders. Finally, down-regulation of MCL-1 in prednisolone-resistant MLL-rearranged leukemia cells by RNA interference, to some extent, led to prednisolone sensitization. Collectively, our findings suggest a potential role for MCL-1 in glucocorticoid resistance in MLL-rearranged infant ALL, but at the same time strongly imply that high-level MCL-1 expression is not the sole mechanism providing resistance to these drugs.

Selective Targeting of CTNBB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs.

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for ß-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.

Glucocorticoid sensitisation in Mixed Lineage Leukaemia-rearranged acute lymphoblastic leukaemia by the pan-BCL-2 family inhibitors gossypol and AT-101.

AIM OF THE STUDY: Resistance to glucocorticoids (GCs) remains a major problem in the treatment of infants with acute lymphoblastic leukaemia (ALL) carrying Mixed Lineage Leukaemia (MLL) translocations. Despite intensive research, the mechanism(s) underlying GC resistance remain poorly understood. Recent studies suggested an important role for the pro-survival BCL-2 family member MCL1 in GC resistance in MLL-rearranged ALL. METHODS: We exposed GC-resistant MLL-rearranged SEMK2 cells to potent MCL1-inhibiting agents, including gossypol, AT-101, rapamycin, SU9516 and obatoclax (GX15-070) and determined GC sensitisation using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays. Using Western blotting we analysed the protein expression of most BCL-2 family members in MLL-rearranged SEMK2 cells after treatment with potent MCL-1 inhibiting agents. RESULTS: Only gossypol and its synthetic analogue AT-101 induced GC sensitivity in MLL-rearranged ALL cells. Remarkably, the GC-sensitising effects of gossypol and AT-101 appeared not to be mediated by down-regulation MCL1 or other anti-apoptotic BCL-2 family members, but rather involved up-regulation of multiple pro-apoptotic BCL-2 family members, in particular that of BIM and BID. CONCLUDING REMARKS: In conclusion, gossypol and AT-101 induce GC sensitivity in MLL-rearranged ALL cells, most likely mediated by the activation of BID and BIM without the necessity to down-regulate anti-apoptotic BCL-2 family members like MCL1. Hence, co-administration of either gossypol or AT-101 during GC treatment of GC-resistant MLL-rearranged ALL patients may overcome GC resistance and improve prognosis in this high-risk childhood leukaemia.