Specific NOTCH1 antibody targets DLL4-induced proliferation, migration, and angiogenesis in NOTCH1-mutated CLL cells

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients

Targeted therapies in CLL. New drugs against CLL recurrent mutations

[eng] Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western countries. It is currently an incurable disease. CLL is a B-lymphoid neoplasm in which the microenvironment plays a key role in the development and evolution of the disease. The complete sequencing of 500 CLL patients described the presence of recurrent mutations, the study of which could improve clinical interventions in the management of this neoplasm and design specific treatments for each patient. In this dissertation we have studied two of the most frequently mutated pathways in CLL: RAS-BRAF-MAPK-ERK and TLR-MYD88, as potential therapeutic targets. The results obtained during this thesis, described below, have been published in two articles published in the journals Haematologica (PMID: 30262568) and Leukemia (PMID: 31197259) in 2019. Patients with mutations in these pathways have differential clinical and biological characteristics. Mutated RAS-BRAF-MAPK-ERKs have higher levels of lactate dehydrogenase, ZAP-70, CD49d, CD38, Trisomy 12 and non-mutated immunoglobulin heavy chain region genes. High percentage of this mutated patients are treated before 5 years after the diagnosis. On the other hand, cases with TLR-MYD88 mutations have a better prognosis. Stimulation of the RAS-BRAF-MAPK-ERK pathway induces an increase in ERK phosphorylation, indicative of increased proliferation and survival. Stimulation of TLR-MYD88 also increases proliferation and survival, but also increases cell migration and cytokine secretion. Thus, the inhibition of these pathways, could be a possible therapeutic strategy for the CLL management. Inhibition of the RAS-BRAF-MAPK-ERK pathway with the BRAF inhibitors already available in the clinic (vemurafenib and dabrafenib) was not possible in cases with mutations in the pathway. In contrast, ulixertinib, a pan-ERK inhibitor, did show positive results, but more studies should be done. Inhibition of the TLR-MYD88 pathway was performed with the IRAK4 (key kinase for the pathway) inhibitor ND2158. Our results demonstrate that ND2158, with a dose-dependent effect, has a preferential effect on CLL cells over B cells from healthy donors. ND2158 treatment in primary cells of CLL shows a reduction in: NF-kB and STAT3-mediated signaling, cytokine secretion, proliferation and migration. We validated its effect in vivo on the Eμ-TCL1 transgenic mouse model, the most widely accepted for the study of CLL and its microenvironment. It has been observed that ND2158 is capable of reducing tumor progression as well as reducing mediated support by the myeloid tumor microenvironment. In contrast, ND2158 inhibits the expansion of effector CD8+ T cells and induces an exhaustion cell phenotype, an unwanted phenomenon for a good efficacy of the drug. To overcome this negative impact we propose a combination with immunotherapy, to improve the function of T lymphocytes and thus preserve the tumor control that the immune system function. We have also shown that ND2158 enhances the effect of two of the currently approved CLL clinical treatments, venetoclax and ibrutinib, thereby opening the door for possible clinical studies based on our data. On the other hand, an in vitro validation project of compounds selected by system biology has also been started. As a result, we have concluded that statins, in combination with current venetoclax or ibrutinib treatments, could also be a new therapeutic strategy for CLL control

Mutations in RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia

Mutations in genes of the RAS-BRAF-MAPK-ERK pathwayhave not been fully explored in patients with chronic lym-phocytic leukemia. We, therefore, analyzed the clinical andbiological characteristics of chronic lymphocytic leukemia patientswith mutations in this pathway and investigated thein vitroresponseof primary cells to BRAF and ERK inhibitors. Putative damaging muta-tions were found in 25 of 452 patients (5.5%). Among these, BRAFwas mutated in nine patients (2.0%), genes upstream of BRAF(KITLG,KIT, PTPN11, GNB1, KRASand NRAS) were mutated in 12 patients(2.6%), and genes downstream of BRAF(MAPK2K1, MAPK2K2, andMAPK1) were mutated in five patients (1.1%). The most frequentmutations were missense, subclonal and mutually exclusive. Patientswith these mutations more frequently had increased lactate dehydro-genase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12and unmutated immunoglobulin heavy-chain variable region genesand had a worse 5-year time to first treatment (hazard ratio 1.8,P=0.025). Gene expression analysis showed upregulation of genes ofthe MAPK pathway in the group carrying RAS-BRAF-MAPK-ERKpathway mutations. The BRAF inhibitors vemurafenib and dabrafenibwere not able to inhibit phosphorylation of ERK, the downstreameffector of the pathway, in primary cells. In contrast, ulixertinib, apan-ERK inhibitor, decreased phospho-ERK levels. In conclusion,although larger series of patients are needed to corroborate these find-ings, our results suggest that the RAS-BRAF-MAPK-ERK pathway isone of the core cellular processes affected by novel mutations inchronic lymphocytic leukemia, is associated with adverse clinical fea-tures and could be pharmacologically inhibited