Uncovering NOTCH1 as a Promising Target in the Treatment of MLL-Rearranged Leukemia

MLL rearrangement (MLLr) is responsible for the development of acute leukemias with poor outcomes. Therefore, new therapeutic approaches are urgently needed. The NOTCH1 pathway plays a critical role in the pathogenesis of many cancers including acute leukemia. Using a CRISPR/Cas9 MLL-AF4/-AF9 translocation model, the newly developed NOTCH1 inhibitor CAD204520 with less toxic side effects allowed us to unravel the impact of NOTCH1 as a pathogenic driver and potential therapeutic target in MLLr leukemia. RNA sequencing (RNA-seq) and RT-qPCR of our MLLr model and MLLr cell lines showed the NOTCH1 pathway was overexpressed and activated. Strikingly, we confirmed this elevated expression level in leukemia patients. We also demonstrated that CAD204520 treatment of MLLr cells significantly reduces NOTCH1 and its target genes as well as NOTCH1 receptor expression. This was not observed with a comparable cytarabine treatment, indicating the specificity of the small molecule. Accordingly, treatment with CAD204520 resulted in dose-dependent reduced proliferation and viability, increased apoptosis, and the induction of cell cycle arrest via the downregulation of MLL and NOTCH1 target genes. In conclusion, our findings uncover the oncogenic relevance of the NOTCH1 pathway in MLLr leukemia. Its inhibition leads to specific anti-leukemic effects and paves the way for further evaluation in clinical settings

Treatment response of advanced HNSCC towards immune checkpoint inhibition is associated with an activated effector memory T cell phenotype

Locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. The introduction of PD-1 inhibitors has led to a significant improvement in survival, but only a subpopulation of patients responds to therapy. Current biomarkers cannot reliably identify these patients. The identification of biomarkers for the prediction and monitoring of immunotherapy is therefore of great importance. In this study, we characterized lymphocyte subsets in the peripheral blood of HNSCC patients under PD-1 inhibition. Patients with primary response (n=11) to PD-1 inhibition showed an increase of the CD3+ effector memory (CD3/EM) population and an elevated expression of the activation marker CD69 in CD3+ T cells, particularly in the CD3/EM subpopulation at 3 months when treatment response was assessed. In contrast, patients with primary treatment failure and progressive disease (n=9) despite PD-1 inhibition had lower absolute lymphocyte counts and an increased expression of CTLA-4 in CD3+ T cells at the time of treatment failure compared with baseline, particularly in CD4+ and CD8+ effector memory populations. Our results demonstrate that HNSCC patients’ response to immune checkpoint inhibition shows a distinct immune signature in peripheral blood, which could help identify refractory patients earlier. Furthermore, strategies to overcome primary therapy failure by inducing a beneficial T cell phenotype or adding alternative immune checkpoint inhibitors could improve response rates and survival of HNSCC patients

The RORɣ/SREBP2 pathway is a master regulator of cholesterol metabolism and serves as potential therapeutic target in t(4;11) leukemia

Dysregulated cholesterol homeostasis promotes tumorigenesis and progression. Therefore, metabolic reprogramming constitutes a new hallmark of cancer. However, until today, only few therapeutic approaches exist to target this pathway due to the often-observed negative feedback induced by agents like statins leading to controversially increased cholesterol synthesis upon inhibition. Sterol regulatory element-binding proteins (SREBPs) are key transcription factors regulating the synthesis of cholesterol and fatty acids. Since SREBP2 is difficult to target, we performed pharmacological inhibition of retinoic acid receptor (RAR)-related orphan receptor gamma (RORγ), which acts upstream of SREBP2 and serves as master regulator of the cholesterol metabolism. This resulted in an inactivated cholesterol-related gene program with significant downregulation of cholesterol biosynthesis. Strikingly, these effects were more pronounced than the effects of fatostatin, a direct SREBP2 inhibitor. Upon RORγ inhibition, RNA sequencing showed strongly increased cholesterol efflux genes leading to leukemic cell death and cell cycle changes in a dose- and time-dependent manner. Combinatorial treatment of t(4;11) cells with the RORγ inhibitor showed additive effects with cytarabine and even strong anti-leukemia synergism with atorvastatin by circumventing the statin-induced feedback. Our results suggest a novel therapeutic strategy to inhibit tumor-specific cholesterol metabolism for the treatment of t(4;11) leukemia

Targeting MYC in combination with epigenetic regulators induces synergistic anti-leukemic effects in MLLr leukemia and simultaneously improves immunity

MLL rearranged (MLLr) leukemias are associated with a poor prognosis and a limited response to conventional therapies. Moreover, chemotherapies result in severe side effects with significant impairment of the immune system. Therefore, the identification of novel treatment strategies is mandatory.Recently, we developed a human MLLr leukemia model by inducing chromosomal rearrangements in CD34+ cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This MLLr model authentically mimics patient leukemic cells and can be used as a platform for novel treatment strategies. RNA sequencing of our model revealed MYC as one of the most important key drivers to promote oncogenesis. However, in clinical trials the BRD4 inhibitor JQ-1 leading to indirect blocking of the MYC pathway shows only modest activity. We and others previously reported that epigenetic drugs targeting MAT2A or PRMT5 promote cell death in MLLr cells. Therefore, we use these drugs in combination with JQ-1 leading to augmented anti-leukemic effects. Moreover, we found activation of T, NK and iNKT cells, release of immunomodulatory cytokines and downregulation of the PD-1/PD-L1 axis upon inhibitor treatment leading to improved cytotoxicity.In summary, the inhibition of MYC and MAT2A or PRMT5 drives robust synergistic anti-leukemic activity in MLLr leukemia. Moreover, the immune system is concomitantly activated upon combinatorial inhibitor treatment, hereby further augmenting the therapeutic efficiency

Targeting MYC in combination with epigenetic regulators induces synergistic anti-leukemic effects in MLLr leukemia and simultaneously improves immunity

MLL rearranged (MLLr) leukemias are associated with a poor prognosis and a limited response to conventional therapies. Moreover, chemotherapies result in severe side effects with significant impairment of the immune system. Therefore, the identification of novel treatment strategies is mandatory. Recently, we developed a human MLLr leukemia model by inducing chromosomal rearrangements in CD34+ cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This MLLr model authentically mimics patient leukemic cells and can be used as a platform for novel treatment strategies. RNA sequencing of our model revealed MYC as one of the most important key drivers to promote oncogenesis. However, in clinical trials the BRD4 inhibitor JQ-1 leading to indirect blocking of the MYC pathway shows only modest activity. We and others previously reported that epigenetic drugs targeting MAT2A or PRMT5 promote cell death in MLLr cells. Therefore, we use these drugs in combination with JQ-1 leading to augmented anti-leukemic effects. Moreover, we found activation of T, NK and iNKT cells, release of immunomodulatory cytokines and downregulation of the PD-1/PD-L1 axis upon inhibitor treatment leading to improved cytotoxicity. In summary, the inhibition of MYC and MAT2A or PRMT5 drives robust synergistic anti-leukemic activity in MLLr leukemia. Moreover, the immune system is concomitantly activated upon combinatorial inhibitor treatment, hereby further augmenting the therapeutic efficiency