Modelling and targeting epigenetic regulators in acute leukemia

A complex network of transcription factors controls self-renewal and differentiation of hematopoietic stem cells (HSC). Mutations or translocations of these epigenetic regulators may result in malignant transformation leading to acute leukemia. A significant fraction of childhood acute myeloid leukemia (AML) patients carry a translocation involving the nuclear receptor-binding SET domain protein 1 (NSD1) histone methyltransferase. To understand its function we ablated the gene in the hematopoietic system of the mouse. Surprisingly, all “Nsd1-null” (Vav1-iCre;Nsd1fl/fl) mice developed a lethal, malignant accumulation of CD71dim/+ TER119- erythroid progenitor cells with aberrant clonogenic activity and impaired terminal maturation of self-renewing erythroblasts in vitro, a phenotype that resembles human acute erythroleukemia. The lack of Nsd1 also reduced the number of HSC starting during fetal liver hematopoiesis. Although gene expression signatures revealed reduced mRNA expression of the erythroid master transcription factor Gata1, erythroblasts of Vav1-iCre;Nsd1fl/fl mice expressed constitutively high levels of GATA1 protein. Interestingly, the cells were significantly impaired in activation but still able to repress several known GATA1 targets. Strikingly, retroviral overexpression of Gata1 induced terminal maturation of Vav1-iCre;Nsd1fl/fl proerythroblasts which was associated with activation of GATA1 target genes. Knockdown of NSD1 in human adult or cord-blood derived CD34+ HSC cells also impaired erythroid differentiation associated with increased protein levels of GATA1. In addition, we found high GATA1 protein levels in several human erythroleukemia cell lines suggesting a key role in aberrant erythroid differentiation. Currently ongoing experiments aim to mechanistically understand Nsd1-mediated GATA1 regulation and erythroid differentiation. Preliminary observations with peptide array-based in vitro methylation assays suggest the possibility for direct methylation of GATA1 by NSD1. We also found aberrant expression of erythroid- associated transcription factor complex members with increased levels of GATA1 and ETO2 but reduced levels of TAL1, E2A and LBD1. Moreover, significant changes in global histone H3K36 methylation were seen in proerythroblasts lacking Nsd1. Taken together, our data so far revealed Nsd1 as a novel regulator of normal and malignant erythropoiesis. Ongoing studies may not only provide mechanistic insights of aberrant transcriptional regulation leading to erythroleukemia but could also set the base to develop novel therapies against this rare but very aggressive disease that is currently incurable in most patients. Next to histone methyltransferases like NSD1, histone acetyltransferases like CBP/p300 are recurrently involved in AML-associated chromosomal translocations and also serve as co-activators of other fusion oncogenes, suggesting therapeutic potential of specific targeting of CBP/p300. We characterized the anti-leukemic potential of I-CBP112, a novel small molecule chemical probe that selectively binds the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. We found that I-CBP112 significantly impaired the clonogenic activity of a series of murine cell lines immortalized by the MLL-CBP fusion and other leukemic fusion oncogenes (MLL-AF9, MLL-ENL, NUP98-HOXA9) in a dose-dependent manner. Similar to the murine cells, we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Likewise, I-CBP112 also reduced colony formation of human primary AML blasts but not of normal CD34+ HSC. Importantly, combination of I-CBP112 with another BRD inhibitor targeting BET proteins (JQ1) or with a chemotherapeutic agent (Doxorubicin) revealed clear synergistic effects on cell survival of the AML cell lines. Extreme limited dilution assays in methylcellulose, as well as bone marrow transplantation experiments revealed that I-CBP112 significantly impaired self-renewal of leukemic stem cells in vitro and reduced the leukemia-initiating potential in vivo. Taken together, we found that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML. In addition to I-CBP112, we tested a pan- bromodomain inhibitor (“bromosporine”, BSP) broadly targeting BRDs including BET. Evaluation of BSP in BET- inhibitor sensitive and non-sensitive leukemic cell-lines revealed strong anti-proliferative activity in semi-solid medium. Similar to treatment with JQ1 (a selective BET inhibitor) BSP arrested in S- cell cycle phase suggesting BET-mediated effects. Finally, non-selective targeting of BRDs by BSP identified BETs as master regulators of primary transcription response in leukemia. Collectively the experiments of this thesis investigated the role of epigenetic regulators in normal and malignant hematopoiesis and explored strategies for selective interference as novel anti-leukemic therapies

Dual targeting of JAK2 and ERK interferes with the myeloproliferative neoplasm clone and enhances therapeutic efficacy.

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients' CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN

Co-Occurring CSF3R W791* Germline and Somatic T618I Driver Mutations Induce Early CNL and Clonal Progression to Mixed Phenotype Acute Leukemia

Chronic neutrophilic leukemia (CNL) relates to mutational CSF3R activation with mem-brane proximal CSF3R mutations such as T618I as driver mutations, but the significance of truncat-ing mutations is not clarified. In CNL, concomitant mutations promote disease progression, but insight into longitudinal acquisition is incomplete. In this study, we investigated the role of co-oc-curring germline and somatic CSF3R mutations in CNL, and assessed the impact of clonal evolution on transformation to acute leukemia. We employed sequential next generation sequencing and SNP array karyotyping to assess clonal evolution in CNL of early manifestation age based on a 33-year-old patient. Germline vs. somatic mutations were differentiated using a sample from the hair folli-cle. To investigate a potential predisposition for CNL development and progression by germline CSF3R-W791*, allelic localizations were evaluated. We detected a somatic CSF3R-T618I mutation at 46% variant allele frequency (VAF) at the time of CNL diagnosis, which co-occurred with a CSF3R-W791* truncation at 50% VAF in the germline. Evaluation of allelic localization revealed CSF3R-T618I and W791* on the same allele. A concomitant ASXL1 mutation at 39% VAF increased to 48% VAF upon transformation to mixed phenotype acute leukemia (MPAL), which has both myeloid and lymphoid features. Clonal evolution further involved expansion of the CSF3R double-mutant clone to 90% VAF via copy neutral loss of heterozygosity on chromosome 1p and the emergence of a RUNX1 mutant subclone. Allogeneic transplantation induced complete remission. This study highlights that CNL not only transforms to AML but also to MPAL. The molecular evolution is especially interesting with a CSF3R-W791* mutation in the germline and acquisition of CSF3R-T618I on the same allele compatible with increased susceptibility for mutation acquisition facilitating RUNX1-related clonal transformation

Nuclear interacting SET domain protein 1 inactivation impairs GATA1-regulated erythroid differentiation and causes erythroleukemia

International audienceThe nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34+ hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1-/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1N1918Q SET-domain mutant induces terminal maturation of Nsd1-/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSDN1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1-/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis

Validation of a Targeted Next-Generation Sequencing Panel for Tumor Mutation Burden Analysis: Results from the Onconetwork Immuno-Oncology Consortium

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r2 = 0.998) and phase 2 (r2 = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research

Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers