ALBERT

Description: MLL rearrangements play a crucial role in leukemogenesis. They are associated with a poor prognosis and new treatment strategies are urgently needed. Here, we used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4/-AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient leukemic cells faithfully mimicking the nature of the disease. Thus, it serves as ideal fundamental basis for pharmacological studies. The catalytic activity of disruptor of telomeric silencing 1-like histone methyltransferase (DOT1L) is critical for the transforming potential of MLL oncogenes. Therefore, inhibition of DOT1L deals as one specific therapeutic approach for MLL-rearranged leukemias currently tested in clinical trials. However, the previous success was limited by restricted response warranting further investigation of DOT1L combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines in vitro and in MLL mouse models. Here, we used DOT1L (EPZ004777) and PRMT5 (EPZ015666) inhibitors alone or in combination in our human MLL-rearranged model demonstrating a dose-dependent reduced proliferation, impairment of cell cycle, upregulation of differentiation marker CD14 and apoptosis measured by flow cytometry and downregulation of known MLL target genes determined by qPCR. Strikingly, the combination of both compounds led to complementary anti-tumor effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: MLL rearranged leukemias are associated with a poor prognosis and response to conventional therapies is limited. Therefore, identification of new targets resulting in the development of novel compounds are urgently needed. However, the lack of authentic model systems due to retroviral MLL fusion protein overexpression and usage of pure mouse models that do not mimic the nature of the original human disease compromise efficient pharmacological studies. We generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system a human MLL-rearranged (MLLr) leukemia model derived from umbilical cord blood cells, that is based on patient specific complete translocations of the MLL, AF4 or AF9 genes and shares phenotypical, morphological and molecular features of patient leukemic cells. Thus, it allows us to reproduce downstream effects caused by MLL fusion proteins on patient-like pathologic levels. Gene set enrichment analysis (GSEA) and expression analysis identified Myc as a critical target in our indefinite in vitro growing human MLLr model. Here, we report the impact of the indirect Myc inhibitor JQ1 on cell proliferation and apoptosis in our authentic human MLLr leukemia model. Methods: We used CRISPR/Cas9 to induce MLL-AF4 and -AF9 translocations in primary hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood. MLLr cells were characterized via RNA sequencing, compared to primary patient cells and subjected to GSEA and differential gene expression analysis. MLLr cells were used to analyze the consequences of the Myc inhibitor JQ1 on proliferation and apoptosis determined by flow cytometry. Results: Patient-specific MLL-AF4 and -AF9 cells were generated with a translocation efficiency of 100% after a cell culture period of 30 days. In liquid culture MLLr cells displayed a distinct survival advantage with indefinite growth potential whereas control cells (HSPCs nucleofected with Cas9 alone) terminally differentiated and eventually died out after 60 days of culture. Our human MLLr model based on complete translocations of the MLL and AF4 or AF9 genes, respectively, shared molecular oncogenic features of patient leukemic cells determined by RNA sequencing: Analysis of differentially expressed genes revealed a typical MLLr leukemia signature, with upregulation of HOXA cluster and downregulation of HOXB cluster. Interestingly, further analysis of the impact of MLL fusion protein on transcriptional programs revealed Myc as a highly important target. Strikingly, treatment with the indirect Myc inhibitor JQ1 resulted in a dose-dependent reduced proliferation (50 nM: 61-65%) and induction of apoptosis (100 nM: 23-46%) of MLLr cells whereas control cells were not affected. Conclusions: Our genome editing approach to generate indefinite growing MLL-AF4 and -AF9 translocated cells faithfully models MLLr leukemia and provides an experimental platform to identify molecular targets and testing of new therapies. Myc inhibitor JQ1 shows robust antiproliferative capacity and promotes cell death serving as a potential new therapeutic target for the treatment of MLLr leukemia. Disclosures No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting potent graft-versus-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients. We showed previously that invariant natural killer T (iNKT) cells promote anti-tumor immunity in murine models of allogeneic HCT without exacerbating graft-versus-host disease (GVHD). We therefore studied iNKT cells in DLIs and investigated how culture-expanded iNKT cells from such DLIs (DLI-iNKTs) could lyse leukemia cells. We analyzed 63 cryopreserved DLI samples by flow cytometry. iNKT cells were identified using the PBS57-loaded CD1d tetramer. Under steady state conditions, iNKT cells represent 0.04% (range, 0.01-0.6) of live donor lymphocytes and need to undergo ex vivo expansion for further experiments and clinical application. We established a two-week protocol resulting in a 300-fold expansion of functional DLI-iNKTs with a purity of 94%. Interestingly, we observed a preferential expansion of CD4+ DLI-iNKTs. This subset turned out to be critical for tolerance induction and prevention of GVHD after allogeneic HCT. For degranulation and leukemia cell lysis assays, culture-expanded DLI-iNKTs were co-cultured with leukemia cells. CD107a as a marker of degranulation and iNKT-cell activity was upregulated on DLI-iNKTs upon engagement with leukemia cells that were subsequently lysed in a dose-dependent manner. We also observed an increased release of cytokines like IFN-γ (85 vs. 7 pg/ml, p=0.04). Moreover, the cytotoxic effects of culture-expanded DLI-iNKTs were CD1d-dependent: blocking the interaction between the MHC-I-like molecule CD1d and the T-cell receptor of DLI-iNKTs abrogated iNKT-cell degranulation and efficient leukemia cell lysis. Our results suggest that iNKT cells from DLIs are involved in anti-tumor immunity after allogeneic HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival. Prior expansion of iNKT cells could promote beneficial effects on tumor control, immune tolerance and overall survival. Disclosures Handgretinger: Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Bethge:Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding; Neovii GmbH: Honoraria, Research Funding.

Description: The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser269 impedes DNA-binding. By genome-engineering, we now introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H)S269A] or a phospho-mimetic [Su(H)S269D] isoform. Su(H)S269D mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H)S269A mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJS221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJS221A rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.