Oncogenesis

Acute myeloid leukemia (AML) is a malignancy of immature myeloid blast cells with stem-like and chemoresistant cells being retained in the bone marrow through CXCL12-CXCR4 signaling. Current CXCR4 inhibitors that mobilize AML cells into the bloodstream have failed to improve patient survival, likely reflecting persistent chemokine receptor localization on target cells. Here we characterize the signaling properties of CXCL12-locked dimer (CXCL12-LD), a bioengineered variant of the naturally occurring oligomer of CXCL12. CXCL12-LD, in contrast to wild-type or locked monomer variants, was unable to induce chemotaxis in AML cells. CXCL12-LD binding to CXCR4 decreased G protein, {beta}-arrestin, and intracellular calcium mobilization signaling pathways, and indicated the locked dimer is a partial agonist of CXCR4. Despite these partial agonist properties, CXCL12-LD increased CXCR4 internalization compared to wildtype and monomeric CXCL12. Analysis of a previously published AML transcriptomic data showed CXCR4 positive AML cells co-express genes involved in survival, proliferation, and maintenance of a blast-like state. The CXCL12-LD partial agonist effectively mobilized stem cells into the bloodstream in mice suggesting a potential role for their use in targeting CXCR4. Together, our results suggest that enhanced internalization by CXCL12-LD partial agonist can avoid pharmacodynamic tolerance and may identify new avenues to better target G protein coupled receptors.

Mutations in the NPM1 gene represent the most common (>30% of patients) genetic alteration in Acute Myeloid Leukaemia (AML) and results in the mis-localisation of the mutated NPM1 protein from a predominantly nucleolar localisation to a predominantly cytoplasmic distribution. Numerous studies of NPM1 mutated AML have focussed on the aberrant cytoplasmic localisation of the mutated protein but efforts to reverse this mis-localisation therapeutically have so far resulted in limited clinical benefit. More recently, attention has shifted towards the nucleus with studies showing that mutant NPM1 binds to specific chromatin regions, where it directly regulates oncogenic gene expression. Here, we use high resolution imaging to demonstrate that Nucleophosmin (NPM1) is critical for maintaining normal nucleoli architecture and specifically the integrity of the nucleoli rim. We report for the first time that NPM1 mutated cell lines and primary samples have aberrant nucleoli architecture and demonstrate that the abnormal nucleoli phenotype is reversible. We also report the novel finding that NPM1 mutated protein forms distinct aggregates in NPM1 mutated cells and characterise these for the first time. This work reveals how nucleolar organisation contributes to the molecular mechanisms underpinning NPM1 driven AML and reveals unexpected novel vulnerabilities to be exploited for therapeutic intervention.

Acute myeloid leukemia (AML) remains the deadliest adult leukemia with dismal clinical outcomes. Since 2020, a combination of BCL-2 inhibitor (venetoclax, VEN) with hypomethylating agent (azacytidine/decitabine, AZA/DAC) has become a new standard of care in elderly or unfit AML patients. However, the underlying mechanism of synergy between venetoclax and azacytidine combination is not well understood. While apoptosis is regarded as the primary mode of cell death mechanism caused by the venetoclax and azacytidine combination, we provide novel evidence for pyroptosis as additional cell death mechanisms in response to venetoclax and azacytidine combination therapy. We found that long-term treatment with azacytidine caused hypomethylation and significant upregulation in DFNA5/GSDME, pore forming Gasdermin family gene that is otherwise silent in myeloid leukemia. We found that azacytidine mediates N-terminal pore-forming DFNA5 cleavage, membrane rupture, and subsequent pyroptosis of DFNA5 overexpressing cells in response to venetoclax and azacytidine. Deletion of DFNA5 reduced total cell viability, where DFNA5 KO cells exclusively underwent apoptosis while DFNA5 OE cells showed increased propidium iodide uptake, a marker for membrane rupture. Overall, our study establishes DFNA5 as an important mediator of venetoclax and azacytidine-induced cell death via non-apoptotic mechanisms.

RationaleMultiple Myeloma (MM) stands as the second most common hematological malignancy characterized by the accumulation of monoclonal plasmocytes within the bone marrow. Despite the introduction of proteasome inhibitors, immunomodulatory agents and CD38-targeting antibodies which have extended survival rates, the disease remains incurable for most patients due to the emergence of resistant clones and frequent relapses. The efficacy of the proteasome inhibitor bortezomib (BTZ) in MM treatment underscores the critical role of the ubiquitin proteasome system (UPS) in this cancer. Deubiquitinases (DUBs), a class of enzymes governing the stability, interactions or localization of cellular proteins by removing ubiquitin modifications, have emerged as promising therapeutic targets across various cancers, including MM. MethodsThrough an exhaustive loss-of-function approach, we have identified for the first time USP39 DUB as a pivotal survival determinant for MM cells. ResultsOur analysis reveals a direct correlation between heightened USP39 mRNA levels and shorter survival in MM patients. Additionally, robust USP39 protein expression is observed in MM patient plasmocytes compared to healthy counterparts. Knockdown of Usp39 not only impedes clonogenic capabilities, but also induces apoptosis, triggers cell cycle arrest and overcomes BTZ resistance. Complementary gain-of-function assays, further elucidate how USP39, by stabilizing the transcription factor ZEB1, enhances the trans-migratory potential of MM cells. ConclusionsIn summary, our findings underscores the pivotal role of the deubiquitinase USP39, suggesting that targeting the USP39/ZEB1 axis hold promise as a prospective diagnostic marker and therapeutic target in MM.

Mutation in FLT3 protein is one of the most common mutations in acute myeloid leukemia (AML). Most patients with FLT3-ITD mutation detected at diagnosis or acquired during treatment display poor prognosis and resistance to tyrosine kinase inhibitors or chemotherapy. Existing clinical and pre-clinical data implicate miR-155 in the carcinogenesis of hematological cancers, including FLT3-assocaited AML. However, the role of miR-155 in regulating FLT3-ITD mutation remains elusive. In this study, we have applied loss-of-function studies using wild-type and mutated leukemic cell line models to validate the functional effect of miR-155 inhibition in leukemic cells. Our bioinformatics analysis indicates that FLT3 has a binding site for miR-155 which makes it a direct target of miR-155. Specific targeting of miR-155 by miR-155 inhibitor induced cell apoptosis and reduced FLT3-ITD-mediated cell proliferation and survival. Our data suggests that miR-155 could be a potential therapeutic target for FLT3-associated AML.

Aggressive subtypes of acute myeloid leukemia (AML) are characterized by increased migratory behavior and poor prognosis prioritizing the need for uncovering relevant mechanisms. While attributed to transcriptional changes, these AMLs manifest dysregulated eIF4E implicating disrupted mRNA metabolism. Here, we observed in AML mouse models, patient specimens, and cell lines that eIF4E drives motility, colonization, engraftment and AML progression. AML cells migrate utilizing Ezrin-positive pseudopods. Unexpectedly, we discovered that eIF4E interacts with Ezrin, that these physically associated factors are required and cooperated to drive an on-demand translation program in pseudopods for motility. Indeed, pseudopods were sites of eIF4E- and Ezrin-dependent translation by implementing the first method to directly mark active ribosomes in situ (Visualizing Translation Activity using RiboLace, VISTA-R). Biochemically, Ezrin bound eIF4E, ribosomal components, and mRNAs consistent with our observed Ezrin-dependent modulation of protein production. This unprecedented physical coupling of motility and translation provisions migratory sites to sustain AML progression. Highlights- eIF4E reduction impairs AML cell motility and disease progression - eIF4E-dependent motility requires Ezrin - Ezrin binds eIF4E, transcripts encoding motility factors and active ribosomes - VISTA-R enabled visualization of active ribosomes and translationally active pseudopods (T-PODs) - T-PODs provide novel on-demand localized translation to sustain mobility at migratory sites Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/707190v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@164f191org.highwire.dtl.DTLVardef@2f8928org.highwire.dtl.DTLVardef@d5ad7forg.highwire.dtl.DTLVardef@719a69_HPS_FORMAT_FIGEXP M_FIG C_FIG

WNT11, a non-canonical WNT ligand, plays well-defined roles in development and tissue architecture; however, its function in cancer remains ambiguous. Here, we characterize WNT11 as a context-dependent suppressor of cancer stemness, invasion, and in vivo tumor formation in human epithelial cancer models. We show that WNT11 upregulation reduces the expression of stemness-promoting genes, suppresses epithelial-to-mesenchymal transition, and inhibits sphere formation and tumor growth. Conversely, downregulation of WNT11 enhances these aggressive malignant properties of cancer cells. Mechanistically, we found that the ability of WNT11 to inhibit RAC1 GTPase activation is essential for its regulation of invasion and self-renewal. In cells unresponsive to WNT11, the connectivity between WNT11 and RAC1 activity is disengaged. Direct manipulation of RAC1 activity in these cells recapitulates the phenotype and molecular signature of WNT11-responsive cells, establishing RAC1 as a critical effector of WNT11-mediated tumor suppression. Taken together, these findings identify the cellular context in which WNT11 suppresses RAC1 activation as a key determinant of its anti-tumor effects and provide a mechanistic framework for understanding the diverse, and sometimes opposing, roles of WNT11 reported in cancer.

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological processes that also exert important roles in disease progression Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the future in vivo.

Competitive interactions between tumor cells and surrounding healthy cells are constantly present during the progression of a solid tumor, and their outcome has been proposed to affect the clinical behavior. Previous studies have described various mechanistic and molecular aspects that characterize this process, overall indicating that cancer cells behave as supercompetitors, which eliminate neighboring healthy cells to gain vital space for growth and infiltration of the tissue. Nevertheless, there is a lack of systematic characterization of these competitive interactions, particularly in the context of cancer in mammals. Furthermore, previous studies in the field of cell competition have primarily focused on homotypic cell competition, involving different clones of the same cell or cells deriving from the same tissue. Data are scarce regarding heterotypic cell competition between two unrelated cell types, which is particularly critical for the understanding of metastatic tumors. In this research, we study cell competition in the context of liver metastases, providing a broad characterization of this process in different relevant scenarios, including cells growing in vitro in 2D and 3D, and in vivo. Results show that in vitro, only a subset of cancer cell lines are coherently strong or moderate competitors against hepatocytes, while the remaining demonstrate poor competitiveness. The competitive proficiency can vary depending on the experimental growth system that is employed, and often predicts the phenotype of liver metastases in terms of aggressiveness and morphology. Finally, our data point towards an involvement of mechanical competition in determining the supercompetitor trait of cancer cells. Altogether, our research provides the first comprehensive characterization of heterotypic cell competition, and indicates that cancer cells possess heterogeneous competitive proficiency towards hepatocytes which can be affected by the growth conditions.

MicroRNAs (miRNA) are small RNAs that function as key modulators of gene expression. Due to their promiscuity of binding, a single miRNA may regulate several genes and hence, multiple pathways simultaneously. In addition, the 3-UTR of mRNA can be recognized by several miRNA for suppression or degradation. We built a microRNA-only Knock-out (miRKo) CRISPR/Cas-9 library to identify essential miRNA in Acute Myeloid Leukemia (AML) using OCI-AML2, OCI-AML3 and U937 cell lines as in vitro models. 10 miRNA were identified to be essential in our screen among all three cell lines: miR-19b-1, -19b-2, 29b-2, -302a, -3678, -3713, -3910-1, -4447, -4718 and -6795. By using weighted degrees of association, we identified pathway hubs that uniquely affect all 3 cell lines by integrating miRNA:mRNA networks using mirDIP and pathway analysis using pathDIP. Through the miRKo screen, network membership analyses and biological anticorrelation scoring through patient data analysis, we identified RRP2CA, RPS6KB-1, CREB1, RPM1A, MAPK10, MAP3K2, ITCH, FBX-W7, NR3C1 and XIAP as likely targets of the essential miRNA in AML; and signal transduction, apoptosis, TGF-beta signalling and MAPK signalling as candidate essential pathways in AML.

Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage with generally poor patient outcomes, owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor NFATC2 is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Further, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA-seq and ChIP-seq, NFATC2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a novel co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/567209v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@10b4dc5org.highwire.dtl.DTLVardef@11ed9aaorg.highwire.dtl.DTLVardef@6457a9org.highwire.dtl.DTLVardef@1b722b4_HPS_FORMAT_FIGEXP M_FIG C_FIG Acute myeloid leukaemia (AML) cells of diverse cytogenetic backgrounds are dependent on NFATC2 for survival; in THP-1 cells, NFATC2 is downstream of the epigenetic regulator KDM4A. NFATC2 promotes G1/S phase transition in the cell cycle and oxidative phosphorylation. In addition, NFATC2 functions downstream of transcription factor MYC, and maintains CCND1 expression.

Interactions with the bone marrow (BM) niche are crucial for promoting self-renewal and survival of acute myeloid leukemia (AML) cells. Consequently, AML cells express a variety of surface receptors to engage with BM niche cells and extracellular matrix proteins, including laminins. Despite the association of laminin receptor expression with stemness in healthy hematopoiesis, the role of laminin receptors in AML remains poorly understood. In this study, we present a comprehensive examination of the laminin receptors integrin 3{beta}1, 6{beta}1, 7{beta}1 and basal cell adhesion molecule (BCAM) in AML. We demonstrate that high mRNA expression of all four laminin receptors correlates with poor overall survival. Notably, integrin 6 and 7 display the highest cell surface presentation among the examined laminin receptors and are higher expressed on AML cells compared to healthy controls. Moreover, our results indicate that integrin 7 expression allows to distinguish between leukemic stem cells (LSC) and non-LSC populations. Specifically, integrin 7 appears to mark non-LSC with enhanced migratory potential. Together, our results confirm the association of high laminin receptor expression with poor prognosis and establish integrin 7 as marker of high migratory non-LSC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/587290v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@bdcc20org.highwire.dtl.DTLVardef@a45fd2org.highwire.dtl.DTLVardef@18f058forg.highwire.dtl.DTLVardef@b613fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

The microRNAs miR-15a and miR-16 are key regulators of the anti-apoptotic oncogene BCL2, playing a significant role in tumorigenesis. These miRNAs function as tumor suppressors by directly targeting BCL2, whose overexpression contributes to cell survival and resistance to therapy in multiple malignancies, including chronic lymphocytic leukemia (CLL). The downregulation or deletion miR-15a/miR-16-1 cluster located on chromosome 13q occurs in about 50% of CLL patients and leads to the overexpression of the oncogenic BCL2, contributing to the survival and proliferation of cancer cells. In this confirmatory study, we provide additional evidence supporting the mechanism by which these miRNAs mediate the inhibition of BCL2 translation, leading to reduced levels of BCL2 protein with no significant effect on BCL2 mRNA degradation. This mechanism has been previously established as a critical pathway in the regulation of apoptosis, particularly in cancer cells where BCL2 overexpression is often associated with resistance to cell death. Our findings reinforce the notion that miRNAs, such as miR-15 and miR-16, bind to the 3-UTR of BCL2 messenger RNA (mRNA), specifically repressing its translation without inducing mRNA degradation. The results from our study align with previous research, confirming that the miRNA-mediated inhibition of BCL2 translation serves as a precise regulatory mechanism that targets protein synthesis rather than mRNA stability. These findings highlight the role of miRNAs in fine-tuning post-transcriptional gene regulation, offering a targeted approach to downregulate oncogenic proteins like BCL2 without disrupting the underlying mRNA, which could be leveraged for more refined therapeutic strategies.

In the past decade, adenosine to inosine (A-to-I) RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes ADAR1 and ADAR2, has been shown to contribute to the development and progression of multiple cancers; however, very little is known about its role in acute myeloid leukemia (AML) - the second most common type of leukemia making up 31% of all adult leukemia cases. Here, we found that ADAR2, but not ADAR1 and ADAR3, is specifically downregulated in core binding factor (CBF) AML with t(8;21) or inv(16). In t(8;21) AML, RUNX1-driven transcription of ADAR2 transcripts was found to be repressed by the RUNX1-ETO fusion protein. Forced overexpression of two ADAR2-regulated RNA editing targets COPA and COG3 indeed inhibits clonogenic growth of human t(8;21) AML cells. Further in vivo animal studies confirmed that ADAR2 could suppress leukemogenesis of t(8;21) AML through its RNA binding and editing capabilities. Our results suggest a novel RNA editing-mediated mechanism leading to t(8,12) AML. Key pointsO_LIADAR2, but not ADAR1 and ADAR3, was specifically downregulated in CBF-AML C_LIO_LIRUNX1-ETO suppresses ADAR2 transcription in t(8;21) AML through binding on its promoter C_LIO_LIRNA editing capability of ADAR2 is essential for its repression of leukemogenesis in an AE9a mouse model C_LI

Patients with Acute Myeloid Leukemia (AML) subtypes, acute erythroleukemia and acute megakaryocytic leukemia (M6 and M7 AMLs, respectively) have a median survival of only a few months with no targeted effective treatment. Our gene expression analysis using the Cancer Cell Line Encyclopedia and CRISPR screen from DepMap showed that M6/M7 AMLs have high levels of the transcription factor GATA1 and depend on GATA1 for survival. While GATA1 was shown to support AML cell proliferation and resistance to chemotherapy, GATA1 has long been considered "undruggable". Here, we identify the small molecule N-(4-hydroxyphenyl)retinamide (4-HPR, Fenretinide) as a novel GATA1 targeting agent in M6 and M7 AML cells, with nM to low M concentrations of 4-HPR causing loss of GATA1. In M6 AML OCIM1 cells, knock-down of GATA1 induced cytotoxicity similarly to low doses 4-HPR while overexpression of GATA1 significantly protected cells from 4-HPR-induced cytotoxicity. In M6 AML cells resistant to current standard-of-care (SOC) Azacytidine plus Venetoclax, 4-HPR synergized with SOC overcoming cell resistance to the drugs. As single-agent, 4-HPR outperformed SOC. In M6 AML cells sensitive to SOC, 4-HPR enhanced and prolonged the growth inhibitory effect of SOC. 4-HPR is a synthetic derivative of vitamin A, and numerous clinical trials have supported its safe profile in cancer patients; therefore, targeted use of 4-HPR against M6 and M7 AMLs may represent a novel therapeutic window. Key Points- Fenretinide (4-HPR) targets the transcription factor GATA1, which was previously thought to be "undruggable" and induces GATA1 loss. - M6 and M7 Acute Myeloid Leukemias (AML) have enriched expression of GATA1 and they can be considered GATA1 positive. - Loss of GATA1 contributes significantly to 4-HPR cytotoxicity in M6 OCIM1 cells. - 4-HPR treatment overcomes chemotherapeutic resistance in M6 Acute Myeloid Leukemia cells, synergizes with standard-of-care and outperforms standard-of-care as a single agent.

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O2) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor ICs were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.

Mesenchymal stromal cells (MSCs) are key components of the tumor microenvironment (TME), influencing leukemia progression through poorly understood mechanisms. We investigated the bioelectrical properties of MSCs derived from pediatric AML patients (AML-MSCs) and identified a significant depolarization of their resting membrane potential (Vmem, -14.7mV) compared to healthy MSCs (h-MSCs, -28.5mV), accompanied by downregulation of CaV1.2 L-type calcium channel expression. AML-MSCs displayed increased spontaneous calcium oscillations, suggesting altered ion homeostasis. Notably, h-MSCs exposed to AML blasts underwent a similar Vmem depolarization (-11.8mV) and CaV1.2 downregulation, indicating that leukemic cells actively reprogram MSCs. Functionally, Vmem depolarization in h-MSCs promoted a pro-leukemic phenotype, whereas hyperpolarization of AML-MSCs restored a normal behavior. CaV1.2 over-expression by lentiviral vectors in AML-MSCs shifted Vmem toward hyperpolarization and partially reversed their leukemia-supportive properties, in part through CaV1.2 transfer via tunneling nanotubes. These findings reveal that AML blasts impose a bioelectrical signature on MSCs, modulating ion channel activity to sustain a leukemic niche. Targeting this electrical reprogramming through CaV1.2 restoration represents a potential strategy to re-establish homeostasis in the bone marrow microenvironment.

Stem/progenitor-like cells are known drivers of tumor progression, chemoresistance, and relapse in Sonic-Hedgehog medulloblastoma (SHH-MB), yet the regulatory mechanisms that sustain these resilient cellular states remain incompletely defined. Here, we identify the RE1-silencing transcription factor (REST) as a key transcriptional regulator that preserves the progenitor compartment in SHH-MB through stabilization of the stemness factor SOX2. Mechanistically, REST activates AKT signaling, which in turn enhances SOX2 protein stability, revealing a REST-AKT-SOX2 axis that supports stem/progenitor identity and ongoing tumor maintenance. Beyond maintaining intrinsic stem-like programs, REST also orchestrates the extrinsic communication network of SHH-MB tumors. Single cell transcriptomic profiling and ligand-receptor interaction mapping highlight Midkine (MDK)-mediated signaling as one of the most upregulated intercellular communication routes in MB. We demonstrate that REST drives this signaling cascade through its control of SOX2. Perturbation of REST or SOX2 results in reduced MDK and its receptor, SDC2 expression, and chromatin immunoprecipitation assays show that SOX2 directly binds and regulates MDK/SDC2 expression, establishing and reinforcing a REST/SOX2 centered transcriptional mechanism that coordinates both progenitor maintenance and cell-cell communication in the malignant compartment. Together, these findings position REST as an integrator of intrinsic progenitor cell programs and extrinsic MDK-mediated signaling in SHH-MB. By linking stemness, communication and potential for therapeutic resistance, the REST-AKT-SOX2-MDK signaling axis emerges as a targetable vulnerability to suppress stem/progenitor driven tumor program in REST-driven SHH-MBs.

Gene fusions are stable protein products often occurring from chromosomal rearrangements. These chimeric proteins typically contain distinct molecular entities from each parent gene, and thus, create a product with altered or aberrant function. Gene fusions are frequently found in cancers, including Leukemia. Here, we characterize the kinase activity and subcellular distribution of the Daple-FLT3 (CCDC88C-FLT3) fusion oncoprotein--a rare, but recurrent gene fusion found in patients with hematological malignancies. The protein contains the FLT3 kinase domain and is activated without ligand stimulation. This leads to activation in STAT5a, AKT, and MAPK signaling, which can be modulated by the tyrosine kinase inhibitors (TKIs) sorafenib, quizartinib, and to a lesser degree, imatinib. Moreover, fusion of this kinase domain to Daple facilitates its localization to the pericentrosomal space and enhances kinase activation. These findings provide evidence that targeting Daple-FLT3 outside of its kinase domain may be a complementary approach with TKI therapy. Key PointsO_LIDaple-FLT3 fusion proteins contain a constitutively active kinase domain, activating distinct signaling molecules in cells C_LIO_LICoiled-coil domain on Daple is dispensable for kinase activation, but necessary for maximal activation C_LI

Myeloid leukemias are frequently associated with pathologically activating mutations in tyrosine kinases [BCR-ABL1 in chronic myeloid leukemia (CML); FLT3 juxtamembrane internal tandem duplication (ITD) mutations, FLT3 and KIT activation loop mutations in acute myeloid leukemia (AML)]. Mutations in these kinases activate RAS, which initiates multiple downstream signaling pathways that regulate cell proliferation, differentiation, and apoptosis. The mechanisms whereby RAS is activated by these kinases is incompletely understood, and a better understanding of the molecular mediators involved in RAS activation may uncover new therapeutic strategies. Here we identify a biologically and therapeutically important novel mechanism whereby BCR-ABL1 and FLT3-ITD activate the critical downstream effector RAS in part through phospholipase C gamma-1 (PLCG1). PLCG1 knockout decreases proliferation of CML and FLT3-ITD-expressing AML cells, reduces RAS nucleotide exchange factor activity, and increases sensitivity of CML cells to BCR-ABL1 tyrosine kinase inhibitors (TKIs). Collectively, these studies suggest that PLCG1 inhibition may augment clinical responses to BCR-ABL1 and FLT3 TKIs in CML and AML.