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.

Nectin stabilization at adherens junctions is counteracted by Rab5a-dependent endocytosis.

Cell-cell junctions undergo constant remodeling, which is crucial for the control of vascular integrity. Indeed, transport of junctional components such as cadherins is understood in increasing depth. However, little is known about the respective pathways regulating localization of nectin at cell-cell junctions. Here, we performed an siRNA-based screen of vesicle regulators of the RabGTPase family, leading to the identification of a novel role for Rab5a in the endocytosis nectin-2 at adherens junctions of primary human endothelial cells (HUVEC). Confocal microscopy experiments revealed disordered nectin-2 localization at adherens junctions upon Rab5a depletion. In addition, internalized nectin-2 was shown to prominently localize to Rab5a-positive vesicles in both fixed and living cells. As shown previously, nectin-2 stabilization at junctions is achieved via drebrin-dependent coupling to the subcortical actin cytoskeleton. Consistently, depletion of drebrin in this study leads to enhanced internalization of nectin-2 from junctions. Strikingly, simultaneous silencing of Rab5a and drebrin restored the junctional localization of nectin-2, pointing to Rab5a as counteracting the drebrin-dependent stabilization of nectin-2 at adherens junctions. This mechanism could be further validated by transendothelial resistance measurements. Collectively, our results identify Rab5a as a key player in the endocytosis of nectin-2 and thus in the regulation of adherens junction integrity in primary human endothelial cells.

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.

TRPM7 controls mesenchymal features of breast cancer cells by tensional regulation of SOX4.

Mechanically induced signaling pathways are important drivers of tumor progression. However, if and how mechanical signals affect metastasis or therapy response remains poorly understood. We previously found that the channel-kinase TRPM7, a regulator of cellular tension implicated in mechano-sensory processes, is required for breast cancer metastasis in vitro and in vivo. Here, we show that TRPM7 contributes to maintaining a mesenchymal phenotype in breast cancer cells by tensional regulation of the EMT transcription factor SOX4. The functional consequences of SOX4 knockdown closely mirror those produced by TRPM7 knockdown. By traction force measurements, we demonstrate that TRPM7 reduces cytoskeletal tension through inhibition of myosin II activity. Moreover, we show that SOX4 expression and downstream mesenchymal markers are inversely regulated by cytoskeletal tension and matrix rigidity. Overall, our results identify SOX4 as a transcription factor that is uniquely sensitive to cellular tension and indicate that TRPM7 may contribute to breast cancer progression by tensional regulation of SOX4.

The TRPM7 interactome defines a cytoskeletal complex linked to neuroblastoma progression.

Neuroblastoma is the second-most common solid tumor in children and originates from poorly differentiated neural crest-derived progenitors. Although most advanced stage metastatic neuroblastoma patients initially respond to treatment, a therapy resistant pool of poorly differentiated cells frequently arises, leading to refractory disease. A lack of insight into the molecular mechanisms that underlie neuroblastoma progression hampers the development of effective new therapies for these patients. Normal neural crest development and maturation is guided by physical interactions between the cell and its surroundings, in addition to soluble factors such as growth factors. This mechanical crosstalk is mediated by actin-based adhesion structures and cell protrusions that probe the cellular environment to modulate migration, proliferation, survival and differentiation. Whereas such signals preserve cellular quiescence in non-malignant cells, perturbed adhesion signaling promotes de-differentiation, uncontrolled cell proliferation, tissue invasion and therapy resistance. We previously reported that high expression levels of the channel-kinase TRPM7, a protein that maintains the progenitor state of embryonic neural crest cells, are closely associated with progenitor-like features of tumor cells, accompanied by extensive cytoskeletal reorganization and adhesion remodeling. To define mechanisms by which TRPM7 may contribute to neuroblastoma progression, we applied a proteomics approach to identify TRPM7 interacting proteins. We show that TRPM7 is part of a large complex of proteins, many of which function in cytoskeletal organization, cell protrusion formation and adhesion dynamics. Expression of a subset of these TRPM7 interacting proteins strongly correlates with neuroblastoma progression in independent neuroblastoma patient datasets. Thus, TRPM7 is part of a large cytoskeletal complex that may affect the malignant potential of tumor cells by regulating actomyosin dynamics and cell-matrix interactions.

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.