The mTOR pathway controls phosphorylation of BRAF at T401.

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

Enzyme Is the Name-Adapter Is the Game.

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein-protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

Comprehensive genetic profiling and molecularly guided treatment for patients with primary CNS tumors.

Despite major advances in molecular profiling and classification of primary brain tumors, personalized treatment remains limited for most patients. Here, we explored the feasibility of individual molecular profiling and the efficacy of biomarker-guided therapy for adult patients with primary brain cancers in the real-world setting within the molecular tumor board Freiburg, Germany. We analyzed genetic profiles, personalized treatment recommendations, and clinical outcomes of 102 patients with 21 brain tumor types. Alterations in the cell cycle, BRAF, and mTOR pathways most frequently led to personalized treatment recommendations. Molecularly informed therapies were recommended in 71% and implemented in 32% of patients with completed molecular diagnostics. The disease control rate following targeted treatment was 50% and the overall response rate was 30%, with a progression-free survival 2/1 ratio of at least 1.3 in 31% of patients. This study highlights the efficacy of molecularly guided treatment and the need for biomarker-stratified trials in brain cancers.

Oncogene-induced TIM-3 ligand expression dictates susceptibility to anti-TIM-3 therapy in mice.

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3 treatment-mediated GVL effects are Tc induced. In contrast to anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti-TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Ab in patients with AML relapse after allo-HCT.

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGFß Expression and Regulatory T-cell Expansion in the Bone Marrow Microenvironment.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFß1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFß antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFß. TGFß1 reduced perforin and TNFα production by T cells in vitro. TGFß1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFß1 expression. In four independent patient cohorts, TGFß1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFß1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFß1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFß antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

DYRK1A signalling synchronizes the mitochondrial import pathways for metabolic rewiring.

Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial protein composition. The TOM complex, the organellar entry gate for mitochondrial precursors in the outer membrane, is a target for cytosolic kinases to modulate protein influx. DYRK1A phosphorylation of the carrier import receptor TOM70 at Ser91 enables its efficient docking and thus transfer of precursor proteins to the TOM complex. Here, we probe TOM70 phosphorylation in molecular detail and find that TOM70 is not a CK2 target nor import receptor for MIC19 as previously suggested. Instead, we identify TOM20 as a MIC19 import receptor and show off-target inhibition of the DYRK1A-TOM70 axis with the clinically used CK2 inhibitor CX4945 which activates TOM20-dependent import pathways. Taken together, modulation of DYRK1A signalling adapts the central mitochondrial protein entry gate via synchronization of TOM70- and TOM20-dependent import pathways for metabolic rewiring. Thus, DYRK1A emerges as a cytosolic surveillance kinase to regulate and fine-tune mitochondrial protein biogenesis.

MOST wanted: navigating the MAPK-OIS-SASP-tumor microenvironment axis in primary pediatric low-grade glioma and preclinical models.

Understanding the molecular and cellular mechanisms driving pediatric low-grade glioma (pLGG)-the most prevalent brain tumor in children-is essential for the identification and evaluation of novel effective treatments. This review explores the intricate relationship between the mitogen-activated protein kinase (MAPK) pathway, oncogene-induced senescence (OIS), the senescence-associated secretory phenotype (SASP), and the tumor microenvironment (TME), integrating these elements into a unified framework termed the MAPK/OIS/SASP/TME (MOST) axis. This integrated approach seeks to deepen our understanding of pLGG and improve therapeutic interventions by examining the MOST axis' critical influence on tumor biology and response to treatment. In this review, we assess the axis' capacity to integrate various biological processes, highlighting new targets for pLGG treatment, and the need for characterized in vitro and in vivo preclinical models recapitulating pLGG's complexity to test targets. The review underscores the need for a comprehensive strategy in pLGG research, positioning the MOST axis as a pivotal approach in understanding pLGG. This comprehensive framework will open promising avenues for patient care and guide future research towards inventive treatment options.

Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines.

INTRODUCTION: Patients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. METHODS: Four patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. RESULTS: Of the tested models, only a BRAFV600E-driven model (BT-40, with additional CDKN2A/Bdel) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro. CONCLUSION: Taken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation.

Dipeptidyl-peptidase 9 regulates the dynamics of tumorigenesis and metastasis in breast cancer.

The cytosolic dipeptidyl-aminopeptidase 9 (DPP9) cleaves protein N-termini post-proline or -alanine. Our analysis of DPP9 mRNA expression from the TCGA 'breast cancer' data set revealed that low/intermediate DPP9 levels are associated with poor overall survival of breast cancer patients. To unravel the impact of DPP9 on breast cancer development and progression, the transgenic MMTV-PyMT mouse model of metastasizing breast cancer was used. In addition, tissue- and time-controlled genetic deletion of DPP9 by the Cre-loxP recombination system was done. Despite a delay of tumor onset, a higher number of lung metastases were measured in DPP9-deficient mice compared to controls. In human mammary epithelial cells with oncogenic RAS pathway activation, DPP9 deficiency delayed tumorigenic transformation and accelerated TGF-ß1 induced epithelial-to-mesenchymal transition (EMT) of spheroids. For further analysis of the mechanism, primary breast tumor cells were isolated from the MMTV-PyMT model. DPP9 deficiency in these cells caused cancer cell migration and invasion accompanied by EMT. In absence of DPP9, the EMT transcription factor ZEB1 was stabilized due to insufficient degradation by the proteasome. In summary, low expression of DPP9 appears to decelerate mammary tumorigenesis but favors EMT and metastasis, which establishes DPP9 as a novel dynamic regulator of breast cancer initiation and progression.

Association of RNF43 Genetic Alterations With BRAFV600E and MSIhigh in Colorectal Cancer.

PURPOSE: Recent studies have provided evidence for a predictive value of RNF43 genetic alterations (GAs) as biomarkers for targeted therapies in microsatellite-stable (MSS) colorectal cancer (CRC). These data have the potential to prioritize treatment strategies in patients with BRAFV600E-mutant CRC and help to identify a subgroup that is more likely to derive benefit versus those patients for whom alternative treatment approaches are needed. We were therefore interested in defining the precise frequency of BRAFV600E and RNF43 GAs and their respective overlap in a large cohort of patients with CRC. METHODS: To address this question, we performed a retrospective analysis that included 52,969 patients diagnosed with CRC from the FoundationCORE database. RESULTS: We observed a striking association of RNF43 GAs with MSI and tumor mutational burden status and BRAFV600E mutations. Overall, 23% of MSS patients with confirmed BRAFV600E mutation harbor an RNF43 GA-which accounts for 1.1% of all patients with CRC and for 15.7% of all CRC BRAFV600E cases. CONCLUSION: Ongoing phase III clinical trials, such as BREAKWATER, should aim to incorporate broader genetic profiling to further validate the superior sensitivity of patients with RNF43-mutant, MSS BRAFV600E CRC to anti-EGFR-/BRAFi-based therapies.

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease.

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1ß, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings.

BRAFΔß3-αC in-frame deletion mutants differ in their dimerization propensity, HSP90 dependence, and druggability.

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAFΔß3-αC oncoproteins usually lack five amino acids in the ß3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAFΔß3-αC oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAFΔLNVTAP>F and two novel mutants, BRAFdelinsFS and BRAFΔLNVT>F, and compare them with other BRAFΔß3-αC oncoproteins. We show that BRAFΔß3-αC oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAFΔß3-αC oncoproteins, e.g., BRAFΔLNVTAP>F, increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAFΔß3-αC mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.

The role of the MDM2/p53 axis in anti-tumor immune responses.

Mouse double minute 2 homolog (MDM2) is a negative regulator of the tumor suppressor p53 and often highly expressed in acute myeloid leukemia (AML) and different solid tumors. Inactivating mutations in TP53, the gene encoding for p53, confers an unfavorable prognosis in AML and increases the risk for relapse after allogeneic hematopoietic cell transplantation (allo-HCT). We review the concept that manipulation of MDM2 and p53 could enhance immunogenicity of AML and solid tumor cells. Additionally, we discuss the mechanisms by which MDM2 and p53 regulate MHC class I and II expression, transcription of dsRNA of endogenous retroviruses, interferon responses, IL-15 production and TRAIL-receptor 1 and 2 expression on malignant cells. The direct effects of MDM2-inhibition or MDM2 deletion in effector T cells are discussed in the context of cancer immunotherapy. The preclinical findings are connected to clinical studies using MDM2-inhibition to enhance anti-tumor immunity in patients. In aggregate, this review summarizes current evidence supporting the use of MDM2-inhibition to restore p53, as well as direct effects of MDM2-inhibition on T cells as an emerging concept for combined anti-tumor immunotherapy against hematological malignancies and beyond.

MAPK inhibitor sensitivity scores predict sensitivity driven by the immune infiltration in pediatric low-grade gliomas.

Pediatric low-grade gliomas (pLGG) show heterogeneous responses to MAPK inhibitors (MAPKi) in clinical trials. Thus, more complex stratification biomarkers are needed to identify patients likely to benefit from MAPKi therapy. Here, we identify MAPK-related genes enriched in MAPKi-sensitive cell lines using the GDSC dataset and apply them to calculate class-specific MAPKi sensitivity scores (MSSs) via single-sample gene set enrichment analysis. The MSSs discriminate MAPKi-sensitive and non-sensitive cells in the GDSC dataset and significantly correlate with response to MAPKi in an independent PDX dataset. The MSSs discern gliomas with varying MAPK alterations and are higher in pLGG compared to other pediatric CNS tumors. Heterogenous MSSs within pLGGs with the same MAPK alteration identify proportions of potentially sensitive patients. The MEKi MSS predicts treatment response in a small set of pLGG patients treated with trametinib. High MSSs correlate with a higher immune cell infiltration, with high expression in the microglia compartment in single-cell RNA sequencing data, while low MSSs correlate with low immune infiltration and increased neuronal score. The MSSs represent predictive tools for the stratification of pLGG patients and should be prospectively validated in clinical trials. Our data supports a role for microglia in the response to MAPKi.

Analysis of RAS and drug induced homo- and heterodimerization of RAF and KSR1 proteins in living cells using split Nanoluc luciferase.

The dimerization of RAF kinases represents a key event in their activation cycle and in RAS/ERK pathway activation. Genetic, biochemical and structural approaches provided key insights into this process defining RAF signaling output and the clinical efficacy of RAF inhibitors (RAFi). However, methods reporting the dynamics of RAF dimerization in living cells and in real time are still in their infancy. Recently, split luciferase systems have been developed for the detection of protein-protein-interactions (PPIs), incl. proof-of-concept studies demonstrating the heterodimerization of the BRAF and RAF1 isoforms. Due to their small size, the Nanoluc luciferase moieties LgBiT and SmBiT, which reconstitute a light emitting holoenzyme upon fusion partner promoted interaction, appear as well-suited to study RAF dimerization. Here, we provide an extensive analysis of the suitability of the Nanoluc system to study the homo- and heterodimerization of BRAF, RAF1 and the related KSR1 pseudokinase. We show that KRASG12V promotes the homo- and heterodimerization of BRAF, while considerable KSR1 homo- and KSR1/BRAF heterodimerization already occurs in the absence of this active GTPase and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. We demonstrate that loss-of-function mutations impairing key steps of the RAF activation cycle can be used as calibrators to gauge the dynamics of heterodimerization. This approach identified the RAS-binding domains and the C-terminal 14-3-3 binding motifs as particularly critical for the reconstitution of RAF mediated LgBiT/SmBiT reconstitution, while the dimer interface was less important for dimerization but essential for downstream signaling. We show for the first time that BRAFV600E, the most common BRAF oncoprotein whose dimerization status is controversially portrayed in the literature, forms homodimers in living cells more efficiently than its wildtype counterpart. Of note, Nanoluc activity reconstituted by BRAFV600E homodimers is highly sensitive to the paradox-breaking RAFi PLX8394, indicating a dynamic and specific PPI. We report the effects of eleven ERK pathway inhibitors on RAF dimerization, incl. third-generation compounds that are less-defined in terms of their dimer promoting abilities. We identify Naporafenib as a potent and long-lasting dimerizer and show that the split Nanoluc approach discriminates between type I, I1/2 and II RAFi. Video Abstract.

Case of a Patient With Pancreatic Cancer With Sporadic Microsatellite Instability Associated With a BRAF Fusion Achieving Excellent Response to Immunotherapy.

In this case report, we discuss a case of pancreatic cancer bearing a BRAF fusion, leading to MAPK activation, MLHph, and finally MSI.

Mast cell deficiency prevents BCR::ABL1 induced splenomegaly and cytokine elevation in a CML mouse model.

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Cre allele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy. Mast cells play a key role in the pro-inflammatory tumor microenvironment of the bone marrow. Shown is a cartoon summarizing our results from the mouse model. BCR::ABL1 transformed MCs, as part of the malignant clone, are essential for the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression.

MDM2 Inhibition Enhances Immune Checkpoint Inhibitor Efficacy by Increasing IL15 and MHC Class II Production.

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction-mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor-treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. IMPLICATIONS: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II-production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti-PD-1 immunotherapy for metastatic melanoma is planned.

Charting co-mutation patterns associated with actionable drivers in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: In recent years, intrahepatic cholangiocarcinoma (iCCA) has evolved as a "role model" for precision oncology in gastrointestinal cancers. However, its rarity, paired with its genomic heterogeneity, challenges the development and evolution of targeted therapies. Interrogating large datasets drives better understanding of the characteristics of molecular subgroups of rare cancers and enables the identification of genomic patterns that remain unrecognized in smaller cohorts. METHODS: We performed a retrospective analysis of 6,130 patients diagnosed with iCCA from the FoundationCORE database who received diagnostic panel sequencing on the FoundationOne platform. Short variants/fusion-rearrangements and copy number alterations in >300 tumor-associated genes were evaluated, and the tumor mutational burden (TMB) as well as the microsatellite instability (MSI) status were available for the majority of the cohort. RESULTS: We provide a highly representative cartography of the genomic landscape of iCCA and outline the co-mutational spectra of seven therapeutically relevant oncogenic driver genes: IDH1/2, FGFR2, ERBB2, BRAF, MDM2, BRCA1/2, MET and KRASG12C. We observed a negative selection of RTK/RAS/ERK pathway co-alterations, and an enrichment of epigenetic modifiers such as ARID1A and BAP1 in patients with IDH1/2 and FGFR2 alterations. RNF43 as well as KMT2D occurred with high frequency in MSIhigh and TMBhigh tumors. CONCLUSION: Detailed knowledge of the most prevalent genomic constellations is key to the development of effective treatment strategies for iCCA. Our study provides a valuable resource that could be used to assess the feasibility of clinical trials and subgroup analyses, spurs the development of translationally relevant preclinical models, and serves as a knowledge base to predict potential mechanisms of resistance to targeted therapies in genomically defined subgroups. IMPACT AND IMPLICATIONS: Due to the high frequency of targetable alterations, molecular diagnostics is recommended in patients with biliary tract cancers, and especially in those with iCCA. The identification of an actionable lesion, however, does not guarantee therapeutic success, and the co-mutational spectrum may act as a critical modifier of drug response. Using a large dataset of comprehensive panel sequencing results from 6,130 patients with iCCA, we provide a detailed analysis of the co-mutational spectrum of the most frequent druggable genetic alterations, which is meant to serve as a reference to establish genetically relevant preclinical models, develop hypothesis-driven combination therapies and identify recurrent genetic profiles.

Degradome-focused RNA interference screens to identify proteases important for breast cancer cell growth.

Proteases are known to promote or impair breast cancer progression and metastasis. However, while a small number of the 588 human and 672 murine protease genes have been extensively studied, others were neglected. For an unbiased functional analysis of all genome-encoded proteases, i.e., the degradome, in breast cancer cell growth, we applied an inducible RNA interference library for protease-focused genetic screens. Importantly, these functional screens were performed in two phenotypically different murine breast cancer cell lines, including one stem cell-like cell line that showed phenotypic plasticity under changed nutrient and oxygen availability. Our unbiased genetic screens identified 252 protease genes involved in breast cancer cell growth that were further restricted to 100 hits by a selection process. Many of those hits were supported by literature, but some proteases were novel in their functional link to breast cancer. Interestingly, we discovered that the environmental conditions influence the degree of breast cancer cell dependency on certain proteases. For example, breast cancer stem cell-like cells were less susceptible to depletion of several mitochondrial proteases in hypoxic conditions. From the 100 hits, nine proteases were functionally validated in murine breast cancer cell lines using individual knockdown constructs, highlighting the high reliability of our screens. Specifically, we focused on mitochondrial processing peptidase (MPP) subunits alpha (Pmpca) and beta (Pmpcb) and discovered that MPP depletion led to a disadvantage in cell growth, which was linked to mitochondrial dysfunction.