ABSTRACT
Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.
Subject(s)
Histone-Lysine N-Methyltransferase , Leukemia, Myeloid, Acute , Methyltransferases , Myeloid-Lymphoid Leukemia Protein , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Proto-Oncogene Proteins , Humans , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Methyltransferases/antagonists & inhibitors , Methyltransferases/genetics , Methyltransferases/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Drug Synergism , Gene Rearrangement , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , MutationABSTRACT
In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.
Subject(s)
CRISPR-Cas Systems , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Gene Library , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Transcription Factors , Cell Line , Antigens, Neoplasm , Neoplasm ProteinsABSTRACT
B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, "multi-layered" genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a "pre-VDJ" fetal cellular origin for both t(4;11) and RAS mut The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs 11.7%, P=0.001), and overall survival (73.7 vs 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia.
Subject(s)
Biomarkers, Tumor , Disease Susceptibility , Genome-Wide Association Study , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/etiology , Biopsy , Bone Marrow/metabolism , Chromosome Aberrations , Gene Expression Profiling , Gene Rearrangement , Genomic Instability , Histone-Lysine N-Methyltransferase/genetics , Homeodomain Proteins/genetics , Humans , In Situ Hybridization, Fluorescence , Mutation , Myeloid-Lymphoid Leukemia Protein/genetics , Phosphatidylinositol 3-Kinases/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Prognosis , Signal Transduction , Survival Analysis , V(D)J Recombination , ras Proteins/metabolismSubject(s)
Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Pyridones/pharmacology , Pyrimidinones/pharmacology , ras Proteins/genetics , Animals , Cell Line, Tumor , Gene Rearrangement , Heterografts , Humans , MAP Kinase Signaling System , Mice , Phosphorylation/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnostic imaging , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Tumor Burden/drug effectsABSTRACT
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.
Subject(s)
Gene Rearrangement , Genes, ras , Mutation , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Age of Onset , Cell Line, Tumor , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 4 , Drug Resistance, Neoplasm/genetics , Gene Expression , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/genetics , Humans , Infant , Mutation Rate , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/mortality , Prognosis , Proto-Oncogene Proteins B-raf/genetics , Translocation, GeneticABSTRACT
In KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive malignancy, oncogenic KMT2A-fusion proteins inappropriately recruit DOT1L to promote leukemogenesis, highlighting DOT1L as an attractive therapeutic target. Unfortunately, treatment with the first-in-class DOT1L inhibitor pinometostat eventually leads to non-responsiveness. To understand this we established acquired pinometostat resistance in pediatric KMT2A::AFF1+ B-ALL cells. Interestingly, these cells became mostly independent of DOT1L-mediated H3K79 methylation, but still relied on the physical presence of DOT1L, HOXA9 and the KMT2A::AFF1 fusion. Moreover, these cells selectively lost the epigenetic regulation and expression of various KMT2A-fusion target genes such as PROM1/CD133, while other KMT2A::AFF1 target genes, including HOXA9 and CDK6 remained unaffected. Concomitantly, these pinometostat-resistant cells showed upregulation of several myeloid-associated genes, including CD33 and LILRB4/CD85k. Taken together, this model comprehensively shows the adaptive potential of KMT2A-rearranged ALL cells upon losing dependency on one of its main oncogenic properties.
ABSTRACT
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.
Subject(s)
Chromosomes, Human/metabolism , Gene Expression Regulation, Leukemic , Myeloid-Lymphoid Leukemia Protein/biosynthesis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Translocation, Genetic , Chromosomes, Human/genetics , Cohort Studies , Female , Gene Expression Profiling , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Humans , Infant , Infant, Newborn , Male , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Recurrence , Risk FactorsABSTRACT
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.
Subject(s)
Drug Resistance, Neoplasm/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prednisone/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Cell Survival/drug effects , Child , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Gene Rearrangement , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Histone-Lysine N-Methyltransferase , Humans , Immunoblotting , Infant , Myeloid Cell Leukemia Sequence 1 Protein , Myeloid-Lymphoid Leukemia Protein/metabolism , Oligonucleotide Array Sequence Analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prednisone/therapeutic use , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Infants with MLL-rearranged infant acute lymphoblastic leukemia (MLL-r iALL) undergo intense therapy to counter a highly aggressive malignancy with survival rates of only 30-40%. The majority of patients initially show therapy response, but in two-thirds of cases the leukemia returns, typically during treatment. The glucocorticoid drug prednisone is established as a major player in the treatment of leukemia and the in vivo response to prednisone monotreatment is currently the best indicator of risk for MLL-r iALL. We used two different single-cell RNA sequencing technologies to analyze the expression of a prednisone-dependent signature, derived from an independent study, in diagnostic bone marrow and peripheral blood biopsies. This allowed us to classify individual leukemic cells as either resistant or sensitive to treatment and show that quantification of these two groups can be used to better predict the occurrence of future relapse in individual patients. This work also sheds light on the nature of the therapy-resistant subpopulation of relapse-initiating cells. Leukemic cells associated with high relapse risk are characterized by basal activation of glucocorticoid response, smaller size, and a quiescent gene expression program with cell stemness properties. These results improve current risk stratification and elucidate leukemic therapy-resistant subpopulations at diagnosis.
Subject(s)
Biomarkers, Tumor/genetics , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Neoplasm Recurrence, Local/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Single-Cell Analysis/methods , Transcriptome , Adult , Child , Child, Preschool , Female , Follow-Up Studies , Gene Expression Regulation, Leukemic , Humans , Infant , Infant, Newborn , Male , Neoplasm Recurrence, Local/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prognosis , Survival Rate , Tumor Cells, CulturedABSTRACT
KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) represents an aggressive type of childhood leukemia characterized by a poor clinical outcome with a survival chance of <50%. Implementing novel therapeutic approaches for these patients is a slow-paced and costly process. Here, we utilized a drug-repurposing strategy to identify potent drugs that could expeditiously be translated into clinical applications. We performed high-throughput screens of various drug libraries, comprising 4191 different (mostly FDA-approved) compounds in primary KMT2A-rearranged infant ALL patient samples (n = 2). The most effective drugs were then tested on non-leukemic whole bone marrow samples (n = 2) to select drugs with a favorable therapeutic index for bone marrow toxicity. The identified agents frequently belonged to several recurrent drug classes, including BCL-2, histone deacetylase, topoisomerase, microtubule, and MDM2/p53 inhibitors, as well as cardiac glycosides and corticosteroids. The in vitro efficacy of these drug classes was successfully validated in additional primary KMT2A-rearranged infant ALL samples (n = 7) and KMT2A-rearranged ALL cell line models (n = 5). Based on literature studies, most of the identified drugs remarkably appeared to lead to activation of p53 signaling. In line with this notion, subsequent experiments showed that forced expression of wild-type p53 in KMT2A-rearranged ALL cells rapidly led to apoptosis induction. We conclude that KMT2A-rearranged infant ALL cells are vulnerable to p53 activation, and that drug-induced p53 activation may represent an essential condition for successful treatment results. Moreover, the present study provides an attractive collection of approved drugs that are highly effective against KMT2A-rearranged infant ALL cells while showing far less toxicity towards non-leukemic bone marrow, urging further (pre)clinical testing.
ABSTRACT
High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.
Subject(s)
Leukemia, Myeloid, Acute , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Glycolysis , Humans , Hypoxia , Leukemia, Myeloid, Acute/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
MLL-rearranged infant acute lymphoblastic leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene expression profile. Here we hypothesized that this characteristic gene expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used differential methylation hybridization to explore the DNA methylation patterns underlying MLL-rearranged ALL in infants. The obtained results were correlated with gene expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wild-type MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4;11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation and effectively induces apoptosis in MLL-rearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLL-rearranged infant ALL cases and guides clinical outcome. Therefore, inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants.
Subject(s)
DNA Methylation , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Translocation, Genetic , Cell Line, Tumor , Cell Survival/drug effects , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 19/genetics , Chromosomes, Human, Pair 4/genetics , Chromosomes, Human, Pair 9/genetics , Cluster Analysis , CpG Islands/genetics , Cytidine/analogs & derivatives , Cytidine/pharmacology , Dose-Response Relationship, Drug , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Humans , Infant , Jurkat Cells , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Survival Analysis , Treatment OutcomeSubject(s)
Gene Expression Regulation, Leukemic , Homeodomain Proteins/genetics , Neoplasm Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Antineoplastic Agents, Hormonal/therapeutic use , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Comparative Genomic Hybridization , DNA Copy Number Variations , Female , Gene Expression Profiling , Homeodomain Proteins/metabolism , Humans , Immunophenotyping , Infant , Infant, Newborn , Male , Mutation , Myeloid Ecotropic Viral Integration Site 1 Protein , Neoplasm Proteins/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prednisone/therapeutic use , Survival Analysis , Treatment OutcomeABSTRACT
Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.
ABSTRACT
B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicted by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged leukemia (MLLr) and non-MLLr iB-ALL leukemia uniformly treated according to the Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression, and gene coexpression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer cell vulnerabilities, revealed a robust iB-ALL-specific gene expression-correlating dmCpG signature, and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient-derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.
Subject(s)
Gene Rearrangement, B-Lymphocyte , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Animals , Cell Proliferation/drug effects , Cell Proliferation/genetics , Core Binding Factor Alpha 2 Subunit/genetics , CpG Islands , DNA Methylation , Epigenesis, Genetic , Epigenome , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Humans , Infant , Mice , Mice, Inbred NOD , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Transcription Factor AP-1/antagonists & inhibitors , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Pediatric MLL-rearranged acute myeloid leukemia (AML) has a generally unfavorable outcome, primarily due to relapse and drug resistance. To overcome these difficulties, new therapeutic agents are urgently needed. Yet, implementing novel drugs for clinical use is a time-consuming, laborious, costly and high-risk process. Therefore, we applied a drug-repositioning strategy by screening drug libraries, comprised of >4000 compounds that are mostly FDA-approved, in a high-throughput format on primary MLL-rearranged AML cells. Here we identified pyrvinium pamoate (pyrvinium) as a novel candidate drug effective against MLL-rearranged AML, eliminating all cell viability at <1000â¯nM. Additional screening of identified drug hits on non-leukemic bone marrow samples, resulted in a decrease in cell viability of â¼50% at 1000â¯nM pyrvinium, suggesting a therapeutic window for targeting leukemic cells specifically. Validation of pyrvinium on an extensive panel of AML cell lines and primary AML samples showed comparable viabilities as the drug screen data, with pyrvinium achieving IC50 values of <80â¯nM in these samples. Remarkably, pyrvinium also induced cell toxicity in primary MLL-AF10+ AML cells, an MLL-rearrangement associated with a poor outcome. While pyrvinium is able to inhibit the Wnt pathway in other diseases, this unlikely explains the efficacy we observed as ß-catenin was not expressed in the AML cells tested. Rather, we show that pyrvinium co-localized with the mitochondrial stain in cells, and hence may act by inhibiting mitochondrial respiration. Overall, this study shows that pyrvinium is highly effective against MLL-rearranged AML in vitro, and therefore represents a novel potential candidate for further studies in MLL-rearranged AML.
ABSTRACT
MLL-rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL-rearranged ALL cells in vitro. Here, we assessed the in vivo anti-leukemic potential of low-dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL-rearranged ALL. Furthermore, we explored whether prolonged exposure to low-dose decitabine could chemo-sensitize MLL-rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic-based and anti-neoplastic compounds. Our data reveal that decitabine prolonged survival in xenograft mice of MLL-rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out-growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low-dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL-rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic-based or anticancer drugs using high-throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line-dependent manner.
ABSTRACT
MLL rearranged and hyperdiploid acute lymphoblastic leukemia (ALL) are characterized by high-level FLT3 expression and constitutive FLT3 activation. As known activating FLT3 mutations are often absent in these patients, we screened the entire FLT3 coding sequence in MLL rearranged and hyperdiploid ALL cases for yet unidentified additional genetic alterations using denaturing D-HPLC. Both in MLL rearranged and hyperdiploid ALL we found that a small minority of samples, 7% and 10% respectively, carried genetic alterations. Although some of these alterations may induce FLT3 activation, the majority of these patients carry wild-type FLT3 genes.
Subject(s)
Myeloid-Lymphoid Leukemia Protein/genetics , Ploidies , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , fms-Like Tyrosine Kinase 3/genetics , Chromatography, High Pressure Liquid , DNA Mutational Analysis , Gene Rearrangement , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/epidemiologyABSTRACT
FLT3 gene mutations have been identified as prognostic factors in myeloid malignancies. Furthermore, FLT3 can be activated by wild type overexpression or ligand-dependent in leukemic cells co-expressing FLT3 ligand (FLT3L). So far no data are available on FLT3/FLT3L expression and activation in JMML. In 51 clinical JMML samples, activating mutations were screened, FLT3 and FLT3L mRNA levels were assessed and the sensitivity of JMML cells to the FLT3 inhibitor PKC412 was tested by MTT assays. No evidence for constitutively activation of FLT3/FLT3L was found in JMML, indicating that FLT3 inhibitors are unlikely to be effective in JMML.
Subject(s)
Leukemia, Myelomonocytic, Juvenile/etiology , fms-Like Tyrosine Kinase 3/physiology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Leukemia, Myelomonocytic, Juvenile/enzymology , RNA, Messenger/analysis , Staurosporine/analogs & derivatives , Staurosporine/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Acute lymphoblastic leukemia (ALL) in infants is an aggressive malignancy with a poor clinical outcome, and is characterized by translocations of the Mixed Lineage Leukemia (MLL) gene. Previously, we identified RAS mutations in 14-24% of infant ALL patients, and showed that the presence of a RAS mutation decreased the survival chances even further. We hypothesized that targeting the RAS signaling pathway could be a therapeutic strategy for RAS-mutant infant ALL patients. Here we show that the MEK inhibitors Trametinib, Selumetinib and MEK162 severely impair primary RAS-mutant MLL-rearranged infant ALL cells in vitro. While all RAS-mutant samples were sensitive to MEK inhibitors, we found both sensitive and resistant samples among RAS-wildtype cases. We confirmed enhanced RAS pathway signaling in RAS-mutant samples, but found no apparent downstream over-activation in the wildtype samples. However, we did confirm that MEK inhibitors reduced p-ERK levels, and induced apoptosis in the RAS-mutant MLL-rearranged ALL cells. Finally, we show that MEK inhibition synergistically enhances prednisolone sensitivity, both in RAS-mutant and RAS-wildtype cells. In conclusion, MEK inhibition represents a promising therapeutic strategy for MLL-rearranged ALL patients harboring RAS mutations, while patients without RAS mutations may benefit through prednisolone sensitization.