CRISPR-Cas9 Library Screening Identifies Novel Molecular Vulnerabilities in KMT2A-Rearranged Acute Lymphoblastic Leukemia.

In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.

High-Throughput Drug Library Screening in Primary KMT2A-Rearranged Infant ALL Cells Favors the Identification of Drug Candidates That Activate P53 Signaling.

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) represents an aggressive type of childhood leukemia characterized by a poor clinical outcome with a survival chance of <50%. Implementing novel therapeutic approaches for these patients is a slow-paced and costly process. Here, we utilized a drug-repurposing strategy to identify potent drugs that could expeditiously be translated into clinical applications. We performed high-throughput screens of various drug libraries, comprising 4191 different (mostly FDA-approved) compounds in primary KMT2A-rearranged infant ALL patient samples (n = 2). The most effective drugs were then tested on non-leukemic whole bone marrow samples (n = 2) to select drugs with a favorable therapeutic index for bone marrow toxicity. The identified agents frequently belonged to several recurrent drug classes, including BCL-2, histone deacetylase, topoisomerase, microtubule, and MDM2/p53 inhibitors, as well as cardiac glycosides and corticosteroids. The in vitro efficacy of these drug classes was successfully validated in additional primary KMT2A-rearranged infant ALL samples (n = 7) and KMT2A-rearranged ALL cell line models (n = 5). Based on literature studies, most of the identified drugs remarkably appeared to lead to activation of p53 signaling. In line with this notion, subsequent experiments showed that forced expression of wild-type p53 in KMT2A-rearranged ALL cells rapidly led to apoptosis induction. We conclude that KMT2A-rearranged infant ALL cells are vulnerable to p53 activation, and that drug-induced p53 activation may represent an essential condition for successful treatment results. Moreover, the present study provides an attractive collection of approved drugs that are highly effective against KMT2A-rearranged infant ALL cells while showing far less toxicity towards non-leukemic bone marrow, urging further (pre)clinical testing.

The transcriptional repressor SNAI2 impairs neuroblastoma differentiation and inhibits response to retinoic acid therapy.

Neuroblastoma is the most common extracranial solid tumor in children and originates from poorly differentiated neural crest progenitors. High-risk neuroblastoma patients frequently present with metastatic disease at diagnosis. Despite intensive treatment, patients often develop refractory disease characterized by poorly differentiated, therapy resistant cells. Although adjuvant therapy using retinoic acid (RA)-induced differentiation may increase event-free survival, in the majority of cases response to RA-therapy is inadequate. Consequently, current research aims to identify novel therapeutic targets that enhance the sensitivity to RA and induce neuroblastoma cell differentiation. The similarities between neural crest development and neuroblastoma progression provide an appealing starting point. During neural crest development the EMT-transcription factor SNAI2 plays an important role in neural crest specification as well as neural crest cell migration and survival. Here, we report that CRISPR/Cas9 mediated deletion as well as shRNA mediated knockdown of the EMT-transcription factor SNAI2 promotes cellular differentiation in a variety of neuroblastoma models. By comparing mRNA expression data from independent patient cohorts, we show that a SNAI2 activity-based gene expression signature significantly correlates with event-free survival. Loss of SNAI2 function reduces self-renewal, 3D invasion as well as metastatic spread in vivo, while strongly sensitizing neuroblastoma cells to RA-induced growth inhibition. Together, our data demonstrate that SNAI2 maintains progenitor-like features in neuroblastoma cells while interfering with RA-induced growth inhibition. We propose that targeting gene regulatory circuits, such as those controlling SNAI2 function, may allow reversion of RA-therapy resistant neuroblastoma cells to a more differentiated and therapy responsive phenotype.

Beyond ion-conduction: Channel-dependent and -independent roles of TRP channels during development and tissue homeostasis.

Transient receptor potential (TRP) channels comprise a family of cation channels implicated in a variety of cellular processes, including proliferation, cell migration and cell survival. As a consequence, members of this ion family play prominent roles during embryonic development, tissue maintenance and cancer progression. Although most TRP channels are non-selective, many cellular responses, mediated by TRP channels, appear to be calcium-dependent. In addition, there is mounting evidence for channel-independent roles for TRP channels. In this review, we will discuss how both these channel-dependent and -independent mechanisms affect cellular programs essential during embryonic development, and how perturbations in these pathways contribute to a variety of pathologies. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.