Selective Vulnerabilities Research Articles

Abstract IA008: Exploiting tumor selective vulnerabilities with structure based drug design

Abstract It is well appreciated that the integrity of both pluripotent and terminally differentiated cellular genomes is under constant pressure from damage provoked by cell-intrinsic and environmental factors. As such, defects in the cellular machinery preserving genomic integrity lead to the accumulation of variants that can promote cancer, but they also confer tumor-cell specific vulnerabilities that present synthetic-lethal therapeutic opportunities. The deepening mechanistic appreciation of tumor-selective pressure on DNA replication-fork fidelity, namely oncogene-induced replication stress, has provoked great interest in the prosecution of drug targets within the cellular machinery that otherwise supports replication fork stability. Poly (ADP-Ribose) glycohydrolase (PARG) is an enzyme with a unique role in the resolution of DNA damage repair and DNA replication fork restart through hydrolysis of Poly (ADP-ribose) (PAR) chains. IDE161 is a potent, selective small molecule inhibitor of PARG that is being developed as an anti-cancer therapeutic for patients with advanced or metastatic cancer harboring defects in homologous recombination repair. In culture, the accumulation of PAR chains upon PARG inhibition causes delayed repair of DNA breaks, replication stress and mitotic catastrophe in settings with collateral liabilities in DNA damage repair and/or replication fork stability. Genome-wide CRISPR-mediated fitness screens across multiple genetic backgrounds revealed strong synthetic lethal interactions of IDE161 with otherwise non-essential components of cellular machinery supporting base excision repair, homologous recombination repair and replication fork stability. In addition, evaluation of IDE161 antiproliferative activity across a panel of 350 molecularly characterized cancer cell lines returned a response profile that was only partially overlapping with that observed with PARPi and was enriched for models harboring vulnerabilities associated with replication stress and/or defects in DNA repair. Notably, many homologous recombination deficient (HRD) cell models display differential dependency on PARG activity vs PARP1/2 activity, suggesting PARG inhibition as a potential therapeutic strategy distinct from PARP inhibitors. In vivo assessment of IDE161 anti-tumor activity in CDX and PDX models mirrored these mechanistic relationships. A number of HRD PDX models displayed a superior response to IDE161 versus PARPi, including robust regressions selectively observed with IDE161 versus niraparib in BRCA2 altered ER+, HER2-breast cancers. Thus, IDE161 has potential as novel targeted therapy that exploits the synthetic lethal relationship between PARG and genomic instability, leading to selective anti-proliferative activity in tumors harboring defects in DNA repair and replication fidelity. Citation Format: Reeja Maskey, Diana Munoz, Megan Conway, Arjun Rao, Vidhya Nagarajan, Ivan Shabalin, Kelly Trego, Jonathan Ryan, Peter Teriete, Christian Frey, Josh Taygerly, Paul Barsanti, Claire Neilan, Jasgit Sachdev, Michael White. Exploiting tumor selective vulnerabilities with structure based drug design [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA008.

Abstract 3063: Defining oncogene-specific metabolic vulnerabilities in a mouse model of hepatocellular carcinoma

Abstract Despite the continued success of advanced cancer therapies, hepatocellular carcinoma (HCC) represents a substantial challenge to current treatment modalities that are not sufficiently effective for the management of advanced disease. Metabolic reprogramming is a hallmark of cancer and many oncogenic signaling pathways impact on cellular metabolism. Here, we are investigating changes in metabolism in different oncogene-driven mouse models of liver cancer to identify selective metabolic vulnerabilities associated with HCC that could be implicated in tumor initiation and progression to be exploited for cancer therapy. Transposon-based vectors coding for different combinations of oncogenes, either Myc and Akt1Myr (CaMIA) or Myc and NrasG12V (CaMIN), were transduced by hydrodynamic tail vein injected into C57BL/6 mice. The resulting liver tumors and control livers were subjected to a comprehensive profiling of polar metabolites (metabolomics) and lipids (lipidomics and total fatty acids) as well as changes in gene expression (transcriptomics). Despite similar overall survival, the two models display marked differences in the presentation of the tumors, with CaMIA showing larger aggressive tumors, while CaMIN livers contain numerous small tumor nodules. Both models also showed evidence for the deregulation of intermediate metabolism, indicating activation of aerobic glycolysis and deregulation of glutamine metabolism. Lipidomic analysis revealed substantial lipidome remodeling in both models. Oncogene-specific alterations included elevated levels of lysophosphatidylcholine (LPC) in CaMIN tumors, while CaMIA tumors displayed high levels of ether phospholipids compared to CaMIN and normal liver. Moreover, the fraction of polyunsaturated fatty acids (PUFAs) was enhanced in lipids from CaMIN tumors, while monounsaturated fatty acids (MUFAs) were enriched in CaMIA tumors. Furthermore, tumors from both genotypes produced higher levels of oxylipins, a class of lipid mediators involved in signaling and inflammation, compared to normal liver. Transcriptome analysis confirmed these metabolic alterations and revealed evidence for enhanced inflammatory signaling in CaMIN. In addition, analysis of tumor microenvironment composition disclosed specific differences in stromal components, indicative of immune evasion through checkpoint activation. Experiments in primary murine liver cancer cell lines confirmed oncogene-specific metabolic rearrangements and suggest elevated sensitivity towards inhibitors of lipid remodeling. Future experiments will investigate the role of altered lipid metabolism in tumor development and immune evasion in liver cancer. Citation Format: Kamal Al-Shami, Marteinn Snaebjornsson, Felix Vogel, Lisa Schlicker, Phillip Pöller, Daniel Dauch, Almut Schulze. Defining oncogene-specific metabolic vulnerabilities in a mouse model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3063.

Abstract B035: A compendium of syngeneic, transplantable pediatric high-grade glioma models reveals subtype-specific therapeutic vulnerabilities

Abstract Pediatric high-grade gliomas (pHGGs) are lethal, incurable brain tumors frequently driven by clonal mutations in histone genes. They often harbor a range of additional genetic alterations that correlate with different ages, anatomical locations, and tumor subtypes. We developed models representing 16 pHGG subtypes driven by different combinations of alterations targeted to specific brain regions. Tumors developed with varying latencies and cell lines derived from these models engrafted in syngeneic, immunocompetent mice with high penetrance. Targeted drug screening revealed unexpected selective vulnerabilities. Moreover, H3.3K27M tumors with PIK3CA, NF1 and FGFR1 mutations were more invasive and harbored distinct additional phenotypes, such as exophytic spread, cranial nerve invasion and spinal dissemination. Collectively, these models reveal that different partner alterations produce distinct effects on pHGG cellular composition, latency, invasiveness, and treatment sensitivity. Citation Format: Michael McNicholas, Antonella De Cola, Zahedeh Bashardanesh, Amelia Foss, Cameron Lloyd, Steven Hebert, Damien Faury, Augusto Faria Andrade, Nada Jabado, Claudia Kleinman, Manav Pathania. A compendium of syngeneic, transplantable pediatric high-grade glioma models reveals subtype-specific therapeutic vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B035.

Computational Discovery and Validation of NAD+ Biosynthesis As Unique Vulnerability in B-Lymphoid Malignancies

Background and Significance: Glucocorticoids have been a mainstay in virtually all therapy regimens for B-lymphoid malignancies since the 1950s. Glucocorticoids selectively kill B-cell leukemia and lymphoma cells but have very limited effects on myeloid and epithelial cells. Likewise, inhibitors of B-cell signaling (e.g. ibrutinib) and selective depletion of B-cells (e.g. rituximab, CD19 CAR-T cells) have achieved important progress and are well tolerated. Drug discovery tool for selective targeting of B-cell malignancies: For these reasons, we developed an interactive computational tool ( lymphoblasts.org) to identify novel B-cell selective vulnerabilities based on integrated genetic and pharmacological compound screens ( Figure 1). Compound screening data from CTD and GDSC databases were reprocessed by fitting 4-parameter log-logistic dose response curves. Differential gene-dependency was also tested using DEMETER2 and Chronos scores, from Project Achilles RNAi and CRISPR datasets, respectively. To prioritize cell-type specific targets, compounds were ranked by average differential rank percentile of both compound-sensitivity and target-gene dependency. NAD+ biosynthesis as unique B-lymphoid vulnerability: Integration of genetic RNAi and CRISPR-based sensitivity data identified NMNAT1 and NAMPT as top-ranking B-cell selective targets that regulate NAD+ salvage and biosynthesis in the nucleus ( Figure 1). NAMPT, was previously identified as “pre-B-cell colony enhancing factor” and functions as rate-limiting enzyme in the salvage biosynthesis pathway of NAD+, which is critical for cellular energy metabolism. These genetic results were further corroborated by the identification of four small molecule inhibitors of NAMPT, with a striking B-cell-selective sensitivity profile. Compared to a panel of 1,078 myeloid leukemia and solid tumor cell lines, B-cell leukemia and lymphoma cell lines (n=98) were dramatically more sensitive to NAMPT inhibitors, including STF31, CAY10618, GMX1778 and Daporinad ( Figure 1). To verify these results based on the DepMap dataset and our drug discovery tool (lymphoblasts.org), we studied the effects of seven NAMPT inhibitors (CAY10618, FK866, GMX1778, OT82, STF118804, STF31 and KPT9274) on a panel of myeloid leukemia and solid tumor cell lines as well as PDX from patients with B-ALL and mantle cell lymphoma. Interestingly, three of these NAMPT inhibitors have demonstrated favorable PK/PD and safety profiles (NCT04281420, NCT04914845, NCT03921879). Genetic modeling of NAMPT-deficiency in B-cell malignancies. To model consequences of Nampt deletion in B-ALL, we transformed pre-B cells from Namptfl/fl mice with BCR-ABL1 and NRAS G12D oncogenes. Deletion of Nampt subverted competitive fitness of B-ALL cells, loss of colony-forming ability and cell cycle arrest. Additionally, we developed a conditional mouse model ( Namptfl/fl x Mb1-Cre) for B-cell-specific Nampt deletion, which resulted near-complete ablation of B-lymphopoiesis. Moreover, in vitro inducible deletion of Nampt caused rapid depletion and reduced colony formation in cultured bone marrow pre-B cells. Deletion of Nampt profoundly affected B-cell energy metabolism, significantly reduced FCCP-stimulated OCR and resulted in cell death owing to energy stress. Metabolomic studies of genetic Nampt-deletion using mass spectrometry revealed depletion of metabolites involved in amino acid metabolism, purine/pyrimidine metabolism, and the TCA cycle. Notably, phosphoserine was among the top-ranked downregulated metabolites, suggesting the involvement of one carbon pathway disruption in mediating energy crisis upon Nampt-deletion in B-ALL cells. Conclusions: These findings highlight that the classical ‘glucocorticoid paradigm’ can be extended to discover novel vulnerabilities, including NAD+ biosynthesis. Computational integration of genetic and pharmacological compound screens was particularly powerful in identifying previously unrecognized opportunities to leverage selective vulnerabilities of B-lymphoid leukemia and lymphoma. Our genetic studies corroborate the unique role of NAMPT and NMNAT1 in B-cell development and metabolic regulation. Further studies will focus on efforts to repurpose three clinically approved NAMPT small molecule inhibitors for the treatment of refractory B-cell malignancies.

MODL-08. A COMPENDIUM OF SYNGENEIC, TRANSPLANTABLE PEDIATRIC HIGH-GRADE GLIOMA MODELS REVEALS SUBTYPE-SPECIFIC THERAPEUTIC VULNERABILITIES

Abstract Pediatric high-grade gliomas (pHGGs) are lethal, incurable brain tumors frequently driven by clonal mutations in histone genes. They often harbor a range of additional genetic alterations that correlate with different ages, anatomical locations, and tumor subtypes. We developed models representing 16 pHGG subtypes driven by different combinations of alterations targeted to specific brain regions. Tumors developed with varying latencies and cell lines derived from these models engrafted in syngeneic, immunocompetent mice with high penetrance. Targeted drug screening revealed unexpected selective vulnerabilities— H3.3G34R/PDGFRAC235Y to FGFR inhibition, H3.3K27M/PDGFRAWT to PDGFRA inhibition, and H3.3K27M/PDGFRAWT and H3.3K27M/PPM1DDC/PIK3CAE545K to combined inhibition of MEK and PIK3CA. Moreover, H3.3K27M tumors with PIK3CA, NF1 and FGFR1 mutations were more invasive and harbored distinct additional phenotypes, such as exophytic spread, cranial nerve invasion and spinal dissemination. Collectively, these models reveal that different partner alterations produce distinct effects on pHGG cellular composition, latency, invasiveness, and treatment sensitivity.

Blocking Mitochondrial Pyruvate Transport Alters Corneal Myofibroblast Phenotype: A New Target for Treating Fibrosis.

The purpose of this study was to critically test the hypothesis that mitochondrial pyruvate carrier (MPC) function is essential for maintenance of the corneal myofibroblast phenotype in vitro and in vivo. Protein and mRNA for canonical profibrotic markers were assessed in cultured cat corneal myofibroblasts generated via transforming growth factor (TGF)-β1 stimulation and treated with either the thiazolidinedione (TZD) troglitazone or the MPC inhibitor alpha-cyano-beta-(1-phenylindol-3-yl) acrylate (UK-5099). RNA sequencing was used to gain insight into signaling modules related to instructive, permissive, or corollary changes in gene expression following treatment. A feline photorefractive keratectomy (PRK) model of corneal wounding was used to test the efficacy of topical troglitazone at reducing α-smooth muscle actin (SMA)-positive staining when applied 2 to 4 weeks postoperatively, during peak fibrosis. Troglitazone caused cultured myofibroblasts to adopt a fibroblast-like phenotype through a noncanonical, peroxisome proliferator-activated receptor (PPAR)-γ-independent mechanism. Direct MPC inhibition using UK-5099 recapitulated this effect, but classic inhibitors of oxidative phosphorylation (OXPHOS) did not. Gene Set Enrichment Analysis (GSEA) of RNA sequencing data converged on energy substrate utilization and the Mitochondrial Permeability Transition pore as key players in myofibroblast maintenance. Finally, troglitazone applied onto an established zone of active fibrosis post-PRK significantly reduced stromal α-SMA expression. Our results provide empirical evidence that metabolic remodeling in myofibroblasts creates selective vulnerabilities beyond simply mitochondrial energy production, and that these are critical for maintenance of the myofibroblast phenotype. For the first time, we provide proof-of-concept data showing that this remodeling can be exploited to treat existing corneal fibrosis via inhibition of the MPC.

Targeting Upregulated cIAP2 in SOX10-Deficient Drug Tolerant Melanoma.

Drug tolerance and minimal residual disease (MRD) are likely to prelude acquired resistance to targeted therapy. Mechanisms that allow persister cells to survive in the presence of targeted therapy are being characterized but selective vulnerabilities for these subpopulations remain uncertain. We identified cellular inhibitor of apoptosis protein 2 (cIAP2) as being highly expressed in SOX10-deficient drug tolerant persister (DTP) melanoma cells. Here, we show that cIAP2 is sufficient to induce tolerance to MEK inhibitors, likely by decreasing the levels of cell death. Mechanistically, cIAP2 is upregulated at the transcript level in SOX10-deficient cells and the AP-1 complex protein, JUND, is required for its expression. Using a patient-derived xenograft model, we demonstrate that treatment with the cIAP1/2 inhibitor, birinapant, during the MRD phase delays the onset of resistance to BRAF inhibitor and MEK inhibitor combination therapy. Together, our data suggest that cIAP2 upregulation in SOX10-deficient subpopulations of melanoma cells induces drug tolerance to MAPK targeting agents and provides a rationale to test a novel therapeutical approach to target MRD.

DIPG-35. IDENTIFYING DRIVER-SPECIFIC VULNERABILITIES IN PAEDIATRIC HIGH GRADE GLIOMA SUBTYPES

Abstract Paediatric high-grade gliomas (pHGGs) are incurable malignant brain tumours and a leading cause of cancer-related death in children. The majority of pHGGs carry lysine-to-methionine (K27M) or glycine-to-arginine/valine (G34R/V) mutations in histone variants H3.1 or H3.3. Moreover, histone mutations associate with different anatomical locations and co-segregating mutations defining distinct tumour subtypes within pHGG. However whether these co-occuring mutations can act as drivers to modify tumour phenotypes and drug sensitivities is currently unknown. In order to functionally evaluate the role of partner alterations and to identify new therapeutic targets, we developed in vivo tumour models of pHGG subtypes using in utero electroporation (IUE) in combination with piggyBac transposon and CRISPR technology. We also established ex vivo glioma stem cell (GSCs) lines from mouse models with different co-segregating mutations, able to engraft in syngeneic immune-competent mice. Then, we performed transcriptome analysis and drug screening identifying selective pharmacological vulnerabilities. Our approach represents a preclinical platform to evaluate subtype-specific precision therapies identifying new pathways involved in brain tumour initiation, progression and maintenance.

Abstract 1175: Systematic profiling of conditional pathway activation as a new class of cancer dependency

Abstract The paradigm of cancer dependencies and targeted therapies has been nearly exclusively focused on inhibiting critical pathways or nodes in cancer. However, conditional activation of signaling pathways as a new source of selective cancer vulnerabilities has not been systematically characterized. In this study, we sought to systematically identify context-specific gene activation lethalities in cancer. We performed lineage-specific mutual-exclusivity (ME) analysis using 69,393 cancer patient genomes in cohorts of AACR-GENIE, TCGA, and Foundation One, across 12 cancer lineages, and identified ME mutation pairs within MAPK, PI3K and WNT pathways indicative of evolutionarily negative selection. To functionally test whether mutual exclusive genetic alterations likely create negative selection pressures in cancer, we developed a lentiviral method to achieve simultaneous ectopic ORF expression ~500 barcoded cancer cell lines for large-scale gain-of-function genetic perturbation. Using this technology, we systematically profiled the pan-cancer vulnerability landscape upon activating 10 key pathway nodes, revealing selective activation dependencies associated with specific biomarkers. We validated the anti-tumor effect of PI3K hyperactivation in PIK3CA/PTEN double-mutant endometrial cancer cell lines. Notably, we find that subsets of APC-mutant colorectal cancers retain active destruction complexes. Upon further depletion of APC or direct expression of β-catenin, the resulting WNT pathway hyperactivation impairs the viability of APC-mutant colorectal cancers in in vitro cell lines and organoids and in in vivo tumor models. Together, our study reveals a new class of conditional gene activation dependencies in cancer. Citation Format: Liang Chang, Nancy Y. Jung, Adel Atari, Devishi Kesar, Diego J. Rodriguez, Matthew A. Rees, Melissa Ronan, William R. Sellers. Systematic profiling of conditional pathway activation as a new class of cancer dependency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1175.

A Compendium of Syngeneic, Transplantable Pediatric High-Grade Glioma Models Reveals Subtype-Specific Therapeutic Vulnerabilities.

Histone-mutant pediatric gliomas are a highly heterogeneous tumor entity. Different histone mutations correlate with different ages of onset, survival outcomes, brain regions, and partner alterations. We have developed models of histone-mutant gliomas that reflect this anatomic and genetic heterogeneity and provide evidence of subtype-specific biology and therapeutic targeting. See related commentary by Lubanszky and Hawkins, p. 1516. This article is highlighted in the In This Issue feature, p. 1501.

Sporadic Creutzfeldt–Jakob disease infected human cerebral organoids retain the original human brain subtype features following transmission to humanized transgenic mice

Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt–Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes.

Human hematopoietic stem cell vulnerability to ferroptosis.

Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.

Abstract PR009: RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition

Abstract Introduction: The RNF43 p.G659fs mutation occurs with a frequency of approximately 8% in colorectal cancer (CRC) and is enriched among microsatellite-instability high tumors. However, despite RNF43 being implicated as a Wnt-signaling tumor suppressor, the function of the RNF43 p.G659fs mutation remains undescribed, with no specific therapies directed against it. We hypothesized that large-scale drug screening coupled with functional validation and testing in CRC pre-clinical models would reveal the functional role and selective vulnerabilities of this alteration, leading to new therapeutic approaches in CRC. Methods: We targeted the RNF43 659 site with CRISPR-Cas9 to generate isogenic RNF43659mut clones of CRC cell lines. We leveraged the Broad Institute’s drug repurposing library of over 5,300 compounds to identify selective hits (z<-3 vs DMSO, >5% selective killing in mutant vs WT) in RNF43659mut isogenic cells. These hits were validated in secondary screens and top drugs were tested in patient-derived organoid and xenograft models of the RNF43 p.G659fs mutation. We assessed the functional role of the mutation using cell viability and colony formation assays, quantitative mass spectrometry proteomics, immunoprecipitation and ubiquitination experiments. The immunomodulatory role of RNF43659mut was assessed through bulk and single-cell RNA Seq analysis of mutant cell lines and patient-derived CRC specimens. Results: Using edited cell line models, we demonstrated that RNF43 p.G659fs has a Wnt-independent oncogenic role in CRC. We performed a comprehensive drug screen in isogenic RNF43659mut HT29 CRC cells with a repurposing library of 5,363 compounds and discovered that RNF43659mut is selectively targeted by PI3K/mTOR inhibitors (p = 1.34 x 10-5, chi-square test). Secondary screens in LS513 RNF43659mut-edited and HCT116 RNF43G659fs-overexpressing cells confirmed these results and we further showed that alpelisib and PF-04691502 (a dual PI3K/mTOR inhibitor) selectively killed patient-derived organoids with the RNF43 p.G659Vfs mutation and reduced tumor growth in RNF43659mut CDX mice (p=0.0005, Student’s t-test). RNF43 p.G659fs increased PI3K/AKT signaling in vitro and in vivo through binding, ubiquitinating and degrading p85 and this was reversed by PI3K/mTOR inhibition, RNF43 knockout or RSPO stimulation. Additionally, in pre-clinical models and single-cell studies of human CRCs (>350,000 single cells), we found that RNF43659mut modulates interferon activity and NK cells in the CRC tumor microenvironment. Conclusions: Through functional and pharmacologic studies in pre-clinical models and human CRCs, we described that RNF43 p.G659fs activates PI3K/AKT signaling and modulates the CRC immune microenvironment. We showed that these effects are reversed with PI3K/mTOR inhibitors and that this class of drugs selectively targets RNF43659mut CRCs. Our results have important therapeutic implications for patients with CRC. Citation Format: Marios Giannakis, Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Xiaozhe Xiong, Carino Gurjao, Maximilien Grandclaudon, Srivatsan Raghavan, Steven M. Corsello, Steven A. Carr, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng. RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR009.

Finding Common Ground on the Site of Onset of Amyotrophic Lateral Sclerosis.

The fundamental origin of amyotrophic lateral sclerosis (ALS) has remained an enigma since its earliest description as a relentlessly progressive degeneration with prominent neuromuscular manifestations that are associated with upper and lower motor neuron dysfunction. Although this remains the hallmark of ALS, a significant proportion of patients will also demonstrate one or more features of frontotemporal dysfunction, including a frontotemporal dementia (FTD). Understanding whether these 2 seemingly disparate syndromes are simply reflective of the co-occurrence of 2 distinct pathologic processes or the clinical manifestations of a common pathophysiologic derangement involving the brain more widely has gripped contemporary ALS researchers. Supporting a commonality of causation, both ALS and FTD show an alteration in the metabolism of TAR DNA-binding protein 43, marked by a shift in nucleocytoplasmic localization alongside a broad range of neuronal cytoplasmic inclusions consisting of pathologic aggregates of RNA-binding proteins. Similarly, several disease-associated or disease-modifying genetic variants that are shared between the 2 disorders suggest shared underlying mechanisms. In both, a prominent glial response has been postulated to contribute to non-cell-autonomous spread. A more contemporary hypothesis, however, suggests that syndromes of cortical and subcortical dysfunction are driven by impairments in discrete neural networks. This postulates that such networks, including networks subserving motor or cognitive function, possess unique and selective vulnerabilities to either single molecular toxicities or combinations thereof. The co-occurrence of one or more network dysfunctions in ALS and FTD is thus a reflection not of unique neuroanatomic correlates but rather of shared molecular vulnerabilities. The basis of such shared vulnerabilities becomes the fulcrum around which the next advances in our understanding of ALS and its possible therapy will develop.

Simple and clear differentiation of spinocerebellar degenerations: Overview of macroscopic and low-power view findings.

Spinocerebellar degenerations (SCDs) are a diverse group of rare and slowly progressive neurological diseases that include spinocerebellar ataxia type 1 (SCA1), SCA2, SCA3, SCA6, SCA7, dentatorubral-pallidoluysian atrophy (DRPLA) and multiple system atrophy (MSA). They are often inherited, and affect the cerebellum and related pathways. The combination of clinical findings and lesion distribution has been the gold-standard for classifying SCDs. This conventional approach has not been very successful in providing a solid framework shared among researchers because their points of views have been quite variable. After identification of genetic abnormalities, classification was overwhelmed by genotyping, replacing the conventional approach far behind. In this review, we describe a stepwise operational approach that we constructed based only on macroscopic findings without microscopy to classify SCDs into three major groups: pure cerebellar type for SCA6 and SCA31; olivopontocerebellar (OPC) type for SCA1, SCA2, SCA7 and MSA; and dentatorubral-pallidoluysian (DRPL) type for SCA1, SCA3, DRPLA and progressive supranuclear palsy (PSP). Spinocerebellar tract involvement distinguishes SCA1 and SCA3 from DRPLA. Degeneration of the internal segment of the pallidum is accentuated in SCA3 and PSP, while degeneration of the external segment is accentuated in SCA1 and DRPLA. These contrasts are helpful in subdividing OPC and DRPL types to predict their genotypes. Lesion distribution represents disease-specific selective vulnerability, which is readily differentiated macroscopically using our stepwise operational approach. Precise prediction of the major genotypes will provide a basis to understand how genetic abnormalities lead to corresponding phenotypes through disease-specific selective vulnerabilities.

Selective vulnerabilities in the proteostasis network of castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is associated with an increased reliance on heat shock protein 70 (HSP70), but it is not clear what other protein homeostasis (proteostasis) factors might be involved. To address this question, we performed functional and synthetic lethal screens in four prostate cancer cell lines. These screens confirmed key roles for HSP70, HSP90, and their co-chaperones, but also suggested that the mitochondrial chaperone, HSP60/HSPD1, is selectively required in CRPC cell lines. Knockdown of HSP60 does not impact the stability of androgen receptor (AR) or its variants; rather, it is associated with loss of mitochondrial spare respiratory capacity, partly owing to increased proton leakage. Finally, transcriptional data revealed a correlation between HSP60 levels and poor survival of prostate cancer patients. These findings suggest that re-wiring of the proteostasis network is associated with CRPC, creating selective vulnerabilities that might be targeted to treat the disease.

Targeting Nutrient Dependency in Cancer Treatment.

Metabolic reprogramming is one of the hallmarks of tumor. Growing evidence suggests metabolic changes that support oncogenic progression may cause selective vulnerabilities that can be exploited for cancer treatment. Increasing demands for certain nutrients under genetic determination or environmental challenge enhance dependency of tumor cells on specific nutrient, which could be therapeutically developed through targeting such nutrient dependency. Various nutrients including several amino acids and glucose have been found to induce dependency in genetic alteration- or context-dependent manners. In this review, we discuss the extensively studied nutrient dependency and the biological mechanisms behind such vulnerabilities. Besides, existing applications and strategies to target nutrient dependency in different cancer types, accompanied with remaining challenges to further exploit these metabolic vulnerabilities to improve cancer therapies, are reviewed.

Single-cell epigenomics reveals mechanisms of human cortical development

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape1. In the developing brain, cell fate specification and topographic identity are important for defining cell identity2 and confer selective vulnerabilities to neurodevelopmental disorders3. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development.

Mitochondrial enzyme GPT2 regulates metabolic mechanisms required for neuron growth and motor function in vivo.

The metabolic needs for postnatal growth of the human nervous system are vast. Recessive loss-of-function mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms in neurons required for neuronal growth and survival. These metabolic processes include neuronal alanine synthesis and anaplerosis, the replenishment of tricarboxylic acid (TCA) cycle intermediates. We performed metabolomics across postnatal development in Gpt2-null mouse brain to identify the trajectory of dysregulated metabolic pathways: alterations in alanine occur earliest; followed by reduced TCA cycle intermediates and reduced pyruvate; followed by elevations in glycolytic intermediates and amino acids. Neuron-specific deletion of GPT2 in mice is sufficient to cause motor abnormalities and death pre-weaning, a phenotype identical to the germline Gpt2-null mouse. Alanine biosynthesis is profoundly impeded in Gpt2-null neurons. Exogenous alanine is necessary for Gpt2-null neuronal survival in vitro but is not needed for Gpt2-null astrocytes. Dietary alanine supplementation in Gpt2-null mice enhances animal survival and improves the metabolic profile of Gpt2-null brain but does not alone appear to correct motor function. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons in vivo. We also observe selective death of lower motor neurons in vivo with worsening motor behavior with age. In conclusion, these studies of the pathophysiology of GPT2 Deficiency have identified metabolic mechanisms that are required for neuronal growth and that potentially underlie selective neuronal vulnerabilities in motor neurons.

IDH Mutations in Glioma: Double-Edged Sword in Clinical Applications?

Discovery of point mutations in the genes encoding isocitrate dehydrogenases (IDH) in gliomas about a decade ago has challenged our view of the role of metabolism in tumor progression and provided a new stratification strategy for malignant gliomas. IDH enzymes catalyze the conversion of isocitrate to alpha-ketoglutarate (α-KG), an intermediate in the citric acid cycle. Specific mutations in the genes encoding IDHs cause neomorphic enzymatic activity that produces D-2-hydroxyglutarate (2-HG) and result in the inhibition of α-KG-dependent enzymes such as histone and DNA demethylases. Thus, chromatin structure and gene expression profiles in IDH-mutant gliomas appear to be different from those in IDH-wildtype gliomas. IDH mutations are highly common in lower grade gliomas (LGG) and secondary glioblastomas, and they are among the earliest genetic events driving tumorigenesis. Therefore, inhibition of mutant IDH enzymes in LGGs is widely accepted as an attractive therapeutic strategy. On the other hand, the metabolic consequences derived from IDH mutations lead to selective vulnerabilities within tumor cells, making them more sensitive to several therapeutic interventions. Therefore, instead of shutting down mutant IDH enzymes, exploiting the selective vulnerabilities caused by them might be another attractive and promising strategy. Here, we review therapeutic options and summarize current preclinical and clinical studies on IDH-mutant gliomas.