SummaryOncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.

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The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukaemia resulting in poor clinical outcomes due to resistance towards chemo- and immuno-therapies. Here we show that the myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate from varying differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex, NuRD. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4-positive cell models indicating that lineage switching in MLL/AF4 leukaemia is driven and maintained by disrupted epigenetic regulation.

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T-cell Acute Lymphoblastic Leukemia (T-ALL) is frequently characterized by glucocorticoid (GC) resistance, which is associated with inferior outcomes, thus highlighting the need for novel therapeutic approaches for GC resistant T-ALL. The pTCR/TCR signaling pathways play a critical role in cell fate decisions during physiological thymocyte development, with an interplay between TCR and glucocorticoid receptor (GR) signaling determining the T-lymphocyte selection process. We performed an shRNA screen in vitro and in vivo in T-ALL cell lines and patient derived xenograft (PDX) samples to identify vulnerabilities in the pTCR/TCR pathway and identified a critical role for the kinase LCK in cell proliferation. LCK knockdown or inhibition with dasatinib (DAS) caused cell cycle arrest. Combination of DAS with dexamethasone (DEX) resulted in significant drug synergy leading to cell death. The efficacy of this drug combination was underscored in a randomized phase II-like murine trial, recapitulating an early phase human clinical trial. T-ALL expansion in immunocompromised mice was significantly impaired using this drug combination, relative to mice receiving control vehicle or single drug treatment, highlighting the immediate clinical relevance of this drug combination for high risk T-ALL patients. Our results thus provide a strategy to improve the efficacy of current chemotherapy platforms and circumvent GC resistance.

Not available.

The regulation of hematopoietic lineage fate and commitment is fundamental to normal and malignant hematopoiesis. Switches between lymphoid and myeloid lineages in leukemia are rare and associated with poor clinical outcome, but potentially very informative regarding the regulation of hematopoietic lineage commitment. In contrast to therapy-related acute leukemia (AL) after a first primary leukemia, lineage-switch ALs arise from a common pre-leukemic or leukemic clone and share a founder mutation, most often rearrangement of MLL at 11q23. The majority of switches are from acute lymphoblastic leukemia (ALL) to acute myeloid leukemia (AML); however, conversions from myeloid to lymphoid and even oscillations between the two lineages have been observed, although the molecular mechanisms underlying lineage switch have not yet been identified. Here we describe a male patient who presented at 9 months of age with a t(4;11)-positive B-ALL and was subsequently treated according to the Interfant06 protocol. He achieved complete remission, but relapsed at the age of 4 years with a t(4;11)-positive AML. He underwent allogeneic BM transplantation and has remained in remission 13 months. Sanger sequencing revealed identical translocation breakpoints in the ALL and AML samples, demonstrating a lymphoid to myeloid lineage switch with a common pre-leukemic or leukaemic cell of origin for both ALs. Interestingly, whereas the AML shows no V(D)J rearrangements, we found incomplete rearrangements in the ALL cells indicating a ProB cell origin. In line with this observation, B-ALL cells expressed 6-fold and 120-fold higher levels of PAX5 and EBF1, respectively, compared to AML blast cells. Microsatellite instability measurements argued against a strong therapy-associated impairment of DNA mismatch repair in the AML. The translocation t(4;11) is the most frequently found chromosomal rearrangement in infant leukaemia and is almost exclusively associated with ALL at presentation, suggesting a strong instructive potential towards the lymphoid cell fate. However, the occurrence of lineage switch in t(4;11) AL demonstrates that this instruction can be overcome by as yet unknown mechanisms. Exome sequencing identified 16 and 98 novel somatic variants in the diagnostic ALL sample (0.23 mutations per Mb) and AML (1.4 mutations per Mb), respectively, of which 10 were shared. Of the total novel somatic mutations, there were 1 and 12 non-synonymous alterations in the B-ALL and AML, respectively, of which one was shared. RNA sequencing confirmed that all 12 genes with non-synonymous mutations were expressed in AML blast cells, and all belonged to the top 25% of expressed genes in both the AML and B-ALL. Genes carrying non-synonymous somatic AML-specific mutations include CHD4 (12p13, NuRD helicase, chromatin maintenance, DNA repair, lineage fidelity, part of MLL complex), NCOA2 (8q13, also known at TIF2 and part of the MOZ/TIF2 fusion gene, cofactor of nuclear receptors including VDR and NR3C1; control of myeloid differentiation, putative tumour suppressor), CEP164 (11q23, centrosome protein involved in microtubule organization, DNA damage response and chromosome segregation) and PPP1R7 (2q37, regulatory subunit of protein phosphatase 1, control of mitosis, regulator of AURKB). All amino acid residues predicted to be mutated are conserved between several species. Each of the identified mutations is located in functionally relevant regions and may, thus, interfere with protein function. Notably, exome and RNAseq showed that all 12 of the AML-specific non-synonymous mutations were found in at least 40% of the reads covering the corresponding genomic positions (the sample analysed constituted 80% blast material), thus suggesting heterozygosity for each mutation and that all mutations are common to the major leukemic clone. Taken together, these data suggest that the B-ALL and AML share a common ancestral pre-leukemic or leukemic cell of origin. Whilst the B-ALL revealed few novel somatic mutations, the change in lineage is associated with the acquisition of a substantial number of novel mutations indicating significant clonal evolution preceding the emergence of myeloid neoplasia. These data identify candidate mutations/genes which may overcome lineage instruction by a leukemic master regulator such as MLL/AF4 and which may therefore play an essential role in the control of hematopoietic lineage fate. Disclosures: No relevant conflicts of interest to declare.

The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.