Cellular Vulnerabilities Research Articles

Defining splicing factor requirements for androgen receptor variant synthesis in advanced prostate cancer.

Resistance to androgen receptor (AR)-targeted therapies represent a major challenge in prostate cancer (PC). A key mechanism of treatment resistance in patients who progress to castrate-resistant PC (CRPC) is the generation of alternatively spliced androgen receptor variants (AR-Vs). Unlike full-length AR (FL-AR) isoforms, AR-Vs are constitutively active and refractory to current receptor-targeting agents hence drive tumour progression. Identifying regulators of AR-V synthesis may therefore provide new therapeutic opportunities in combination with conventional AR-targeting agents. Our understanding of AR transcript splicing, and the factors that control the synthesis of AR-Vs, remains limited. While candidate-based approaches have identified a small number of AR-V splicing regulators, an unbiased analysis of splicing factors important for AR-V generation is required to fill an important knowledge gap and furnish the field with novel and tractable targets for PC treatment. To that end, we conducted a bespoke CRISPR screen to profile splicing factor requirements for AR-V synthesis. MFAP1 and CWC22 were shown to be required for the generation of AR-V mRNA transcripts and their depletion resulted in reduced AR-V protein abundance and cell proliferation in several CRPC models. Global transcriptomic analysis of MFAP1-depleted cells revealed both AR-dependent and -independent transcriptional impact, including genes associated with DDR. As such, MFAP1 downregulation sensitised PC cells to ionising radiation suggesting therapeutically targeting AR-V splicing could provide novel cellular vulnerabilities which can be exploited in CRPC. Implications: We have utilised a CRISPR screening approach to identify key regulators of pathogenic AR splicing in prostate cancer.

Neurotoxic mechanisms of dexamethasone in SH-SY5Y neuroblastoma cells: Insights into bioenergetics, oxidative stress, and apoptosis

Despite the known therapeutic uses of dexamethasone (DEX), the specific mechanisms underlying its neurotoxic effects in neuronal cells, particularly in undifferentiated human neuroblastoma (SH-SY5Y) cells, remain inadequately understood. This study aims to elucidate these mechanisms, emphasizing bioenergetics, oxidative stress, and apoptosis, thereby providing novel insights into the cellular vulnerabilities induced by chronic DEX exposure. The findings revealed significant reductions in cell viability, altered membrane integrity with LDH leakage, decreased intracellular ATP production, and the electron transport chain complexes I and III activity inhibition. DEX significantly increased the release of the reactive species and peroxidation of lipids, as well as of Nrf2 expression. At the same time, it simultaneously led to a decline in the activities of the antioxidant catalase and superoxide dismutase enzymes, along with a depletion of glutathione reserves. The apoptosis process was exhibited by a significant elevation of caspases 3 and 8 activities with overexpression of mRNA BAX, inhibition of BCL-2, and a significant upregulation of the BAX/BCL-2 ratio. Assessment of neuronal development genes (GAP43, CAMK2A, CAMK2B, TUBB3, and Wnts) by quantitative PCR assay showed increased expression of CAMK2A, CAMK2B, and Wnt3a with a significant reduction in GAP43 mRNA levels. Collectively, this study proved that DEX was cytotoxic to SH-SY5Y via bioenergetic disruption, mitochondrial dysfunction, oxidative stress, and apoptosis.

Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage

Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.

Up-and-Coming Drugs for the Treatment of Vitiligo.

Vitiligo is a chronic autoimmune disease that causes depigmented patches on the skin. It affects 0.5%-2.0% of the global population. It goes beyond physical appearance, often leading to stigmatization, low self-esteem, and depression, burdening patients with psychosocial challenges. The pathogenesis of vitiligo involves the loss of melanocytes due to autoreactive CD8+ T cells, triggered by environmental stressors and exacerbated by cellular vulnerabilities and immune responses. The release of danger signals and pro-inflammatory factors initiates an immune cascade perpetuating melanocyte destruction, mainly driven by interferon-γ and the C-X-C motif chemokine ligand 9/10-chemokine receptor 3 axis. Long-lasting tissue-resident memory T cells (Trms) and cytokines contribute to lesion persistence. Current treatments focus on topical steroids and tacrolimus, systemic steroids, and phototherapies, but their efficacy remains suboptimal, necessitating the development of new therapeutic options. Building on recent advancements in understanding the immunological mechanisms in vitiligo pathogenesis, with the initiation of Food and Drug Administration approval of topical ruxolitinib, various potential treatment options such as JAK inhibitors, cytokine blockers, and Trm or regulatory T cell targeting agents are being clinically researched and anticipated for vitiligo based on both preclinical and clinical data. This review aims to categorize and summarize the diverse investigational drugs currently undergoing clinical trials for vitiligo. By examining clinical outcomes, it is anticipated that this review will bring hope to dermatologists and patients regarding vitiligo, a condition that has historically posed challenges and transform it into a realm of potential possibilities.

Mitochondrial Functional Profiling Links Apoptotic Sensitivity to Treatment Outcomes Based on Age and Cell Lineage in Acute Leukemia

Background Survival outcomes in paediatric T and B cell acute lymphoblastic leukemia (ALL) are far superior to acute myeloid leukemia (AML). In addition, children with ALL and AML have better response rates than adults. While the basis for these differences in therapy outcomes is multifactorial, it is poorly understood. The majority of anticancer therapies activate apoptosis signalling to target cancer cells. The balance between pro-survival (BCL-2, BCL-XL and MCL-1) and pro-apoptotic proteins as well as evasion of apoptosis, plays a key role in cancers. Dependencies to these anti-apoptotic pro-survival proteins is a hallmark of haematopoietic malignancies and can be exploited with novel agents especially by using Venetoclax (BCL-2 inhibitor) and Navitoclax (BCL-2 and BCL-XL inhibitor) that are now available in the clinic. We hypothesized that difference in apoptosis threshold (priming) based on cell lineage and patient age offers key functional insights into the differences in outcomes as well as gives us an in vitro window to assess inherent BCL-2 or BCL-XL dependencies that can be targeted. Methods We prospectively and retrospectively examined functional regulation of apoptosis by performing a BH3 profiling assay on leukemia patient specimens from two tertiary cancer centres in Singapore. Since BCL-2 family consists of different anti-apoptotic proteins, we asked whether protection from specific pro-survival BCL-2 family proteins such as BCL-2, BCL-XL or MCL-1, predicted by sensitivity to BAD, HRK and MS-1 peptide, respectively, contribute to overall difference in apoptotic sensitivity between lymphoid and myeloid precursors. We also aimed to determine dominant factors that govern differences in clinical chemosensitivity to lymphoid compared to myeloid tumours. Blasts isolated via flow cytometry from bone-marrow aspirate (BMA) or peripheral blood were exposed to a variety of pro-apoptotic BH3 peptides to measure mitochondrial outer membrane permeabilization (cytochrome c release). The percentage of cytochrome c loss with each BH3 peptide at different concentrations was normalized to the cytochrome c release from negative control DMSO wells. Assays were performed in triplicate. Alamethacin was used as positive control. Flow cytometric data was acquired with a BD analyser, analysed with FlowJo and plotted in GraphPad Prism. Results Initial analysis of seventy one patient samples are presented here (40 AML, 17 T-ALL, 12 B-ALL and 2 MPAL). Lymphoblasts showed significantly increased apoptotic sensitivity as measured by Bim response compared to myeloblasts (p<0.001). We found lymphoblasts to be significantly more dependent on BCL-2 and BCL-XL compared to myeloblasts (p<0.001). Higher MCL-1 dependence was also noted in B-ALL cells as compared to AML (p<0.05). We next asked if apoptotic sensitivity measured by mitochondrial priming could explain higher chemosensitivity of pediatric over adult patients. Indeed we noted increase in overall priming in paediatric (N=26) as compared to adults (N=36) irrespective of cell of origin (P<0.001). Within the AML cohort, we found pediatric AML (N=8) to be more sensitive to Bim peptide compared to adults (N=32), as well as showed closer proximity to apoptosis (p<0.05). Of note there were no differences in overall priming or selective dependencies between diagnosis and relapse/refractory (R/R) patients in AML or ALL cohorts. By using blasts of AML and +ALL patients (N=15), we found that increased mitochondrial priming response to HRK+MS1 peptides (which infer BCL-XL and MCL-1 dependence respectively) inversely correlated with achievement of remission with venetoclax based therapy (p<0.001). Mitochondrial sensitivity to MS-1 peptide, increased from pre-treatment to on treatment to relapse (N=3/3 patients) in sequential samples for three patients suggesting an opportunity for the use of MCL-1 inhibition in relapse setting. Conclusion Our study shows increased overall priming in lymphoblast as compared with myeloblast and in pediatric as compared to blasts from adult leukaemia specimens. These findings offer a functional window to further explain differences in outcomes in leukaemia in different ages as well as lineages. Innovative trial designs incorporating cellular vulnerabilities to anti-apoptotic proteins using functional BH3 profiling as an accessory biomarker will enable improved treatment outcomes with reduced toxicity.

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

P53-regulated lncRNAs in cancers: from proliferation and metastasis to therapy.

Long non-coding RNAs (lncRNAs) have been identified as master gene regulators through various mechanisms such as transcription, translation, protein modification and RNA-protein complexes. LncRNA dysregulation is frequently associated with a variety of biological functions and human diseases including cancer. The p53 network is a key tumor-suppressive mechanism that transcriptionally activates target genes to suppress cellular proliferation in human malignancies. Recent research indicates that lncRNAs play an important role in the p53 signaling pathway. In this review, we summarize the current knowledge of lncRNAs in p53-relevant functions and provide an overview of how these altered lncRNAs contribute to tumor initiation and progression. We also discuss the association between lncRNA and up- or downstream genes of p53. These findings imply that lncRNAs can help identify cellular vulnerabilities that may prove to be promising potential biomarkers and therapeutic targets for cancer treatment.

Chemical Proteomics with Novel Fully Functionalized Fragments and Stringent Target Prioritization Identifies the Glutathione-Dependent Isomerase GSTZ1 as a Lung Cancer Target.

Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of 20 fragment-based probes containing natural product-based privileged structural motifs for small-molecule lead discovery. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via "click chemistry," and subsequent target identification through label-free quantitative liquid chromatography-tandem mass spectrometry analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S-transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing, and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory non-small cell lung cancer cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for the identification of unique target candidates, such as GSTZ1 in refractory lung cancer.

Gene Editing and Multi-Omics Reveal Novel Metabolic Vulnerabilities in t(4;14)-Associated Multiple Myeloma

Background: Chromosomal translocation t(4;14) is a recurrent genetic lesion in multiple myeloma (MM) which drives aberrant overexpression of the histone methyltransferase NSD2 and is associated with a more guarded prognosis. Despite its oncogenic outcomes, NSD2 overexpression in MM cells could simultaneously confer cellular vulnerabilities that can be therapeutically exploited. Aim: To define potential therapeutic susceptibilities driven by NSD2 overexpression in t(4;14) MM. Methods: We performed genome-wide knockout screening in isogenic NSD2 high and low KMS11 cells using the Brunello CRISPR library, followed by validation of key target genes in several different t(4;14) MM cell lines. Results: These screens revealed increased reliance of NSD2-overexpressing cells on multiple cellular processes including mitochondrial respiration, transcriptional elongation and proteasomal degradation. One of the top preferential dependencies in NSD2-high MM cells is the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). AK2 catalyzes the reversible interconversion of ADP to ATP and AMP. Cancer Dependency Map (depmap) analysis indicated that AK2 loss is detrimental to most MM cell lines but it is not a dependency in most solid tumor cells. Reliance on AK2 was modest in t(4;14) MM cells engineered to express low levels of NSD2 but the detrimental effect of AK2 depletion by shRNA or CRISPR was exacerbated upon enforced overexpression of NSD2. Gene expression studies showed that AK2 suppression leads to induction of genes involved in oxidative stress response. Metabolomic profiling of NSD2 high and low isogenic MM cells demonstrated striking reduction of NAD+ and elevation of nicotinamide (NAM) levels in NSD2 overexpressing cells, which correlates with a 10-fold induction of expression of the NAD+ consuming enzyme CD38 by NSD2. NAD+ and its metabolite NADP+ are essential co-factors of multiple antioxidant enzymes and its depletion can exacerbate oxidative damage. Therefore, NAD+ depletion likely accounts for the increased susceptibility of NSD2 high MM cells to AK2 loss. In vitro cell proliferation/viability assays demonstrated increased sensitivity of NSD2 high MM cells to PI exposure suggesting that NSD2 overexpression drives a higher load of unfolded proteins, correlating with loss of NAD+ and redox system activity. AK2 suppression in MM cells also impairs activation of the adaptive unfolded protein response through the IRE1a-XBP1 axis leading to decreased protein folding capacity. The combination of high unfolded protein load and poor ATP shuttling to the endoplasmic reticulum (ER) secondary to AK2 loss may represent an additional mechanism of selective lethality. Furthermore, AK2 deficient MM cells displayed increased sensitivity to PIs. Transcriptomic analyses in NSD2 high and low MM cells revealed that NSD2 overexpression, besides driving an oncogenic transcriptional program, alters the expression of several genes involved in nucleotide metabolism. Perturbed nucleotide metabolism was further documented by untargeted metabolite profiling which showed a notable depletion of several pyrimidine metabolites when NSD2 is overexpressed. Intriguingly, induced reduction of pyrimidine synthesis by treatment with DHODH inhibitors appeared to be more detrimental in NSD2 overexpressing MM cells. Conclusions: NSD2 overexpression in MM cells alters cellular metabolism and confers higher susceptibility to oxidative stress and unfolded protein loads. By inducing the expression of NAD+ consuming enzymes, NSD2 overexpression results in NAD+ depletion which likely underlies the described vulnerabilities. Loss of the mitochondrial adenine nucleotide regulator AK2 is more detrimental to NSD2 overexpressing MM cells as it diminishes efficient ATP utilization in the ER perturbing the ability of MM cells to handle high loads of unfolded immunoglobulins and mitigate oxidative stress. Furthermore, integrated analyses of the different omics datasets highlighted other NSD2-driven liabilities, including mitochondrial respiration, transcriptional elongation and nucleotide metabolism, that can be targeted to improve prognosis of MM.

Stimulation of autophagy and synaptic maintenance are commonalities induced by an exercise‐mimetic and diet supplement to avoid initiators of age‐related cognitive decline

Brain aging causes synaptic and cellular vulnerabilities, leading to cognitive impairment and an increased risk for dementia. Removal of old and damaged proteins becomes less efficient with age, causing protein accumulation stress and corresponding synaptopathy as protein clearance systems fail. Interestingly, physical exercise and a healthy diet are well known to promote cognitive health, perhaps by delaying or reducing age‐related changes that cause synaptic compromise. It is vital to understand the mechanism by which exercise and diet positively influence brain health since they are the only avenues implicated in reducing dementia risk. Accordingly, we tested if an exercise‐mimetic and a dietary supplement have a common influence on the protein clearing autophagy‐lysosomal pathway, and if such a mechanistic link promotes synaptic maintenance and resilience. Our study used long‐term brain explants that exhibit multi‐neural cell types and subfields, as well as native neuronal circuitries and synaptic density as compared to the intact adult brain. The exercise‐mimetic compound β‐guanidinopropionic acid (β‐GPA), a creatine analog, and a specific extract of American ginseng (P. quinquefolius; USANA Health Sci), were applied daily to the matured, 3‐dimensional cultures of hippocampal tissue for 3‐6 days. The gently harvested explants were then assessed by immunoblot for several markers including the cathepsin B (CatB) component of the autophagy‐lysosomal pathway, a protease found to improve clearance of pathogenic proteins and preserve synaptic and cognitive measures in models of AD, α‐synucleinopathy, and mild cognitive impairment (Mueller‐Steiner et al. 2006 ‐ Neuron 51:703; Butler et al. 2011 ‐ PLoS ONE 6:e20501; Hwang et al. 2019 ‐ Inter J Mol Sci 20:4432). Both β‐GPA and the American ginseng extract improved the levels of the active CatB isoform and increased autophagic flux. In addition, they both had a positive influence on synaptic markers. Next, β‐GPA and American ginseng were tested in groups of explant cultures that were exposed to a lysosomal inhibitor (chloroquine) as a model for the age‐related compromise of lysosomal protein clearance capacity. Again, both β‐GPA‐ and American ginseng‐treated hippocampal explants displayed protection of pre‐ and postsynaptic proteins, similar to the protected levels produced through pharmacological enhancement of CatB using small‐molecule modulators with drug‐like properties (Viswanathan et al. 2012 – ACS Med Chem Lett 3:920). The significant results from the chloroquine model indicate that preventing age‐related lysosomal stress can slow or delay the associated synaptopathy by different strategies that may be additive. Together, the different experiments support the idea that exercise, exercise‐mimetics, and certain natural products help to limit age‐related synaptic deterioration that is known to be the best correlate of cognitive failure during aging and dementia. The common protective influences mediated by the exercise‐mimetic and the dietary supplement point to protein clearance as having a mechanistic role for healthy cognitive aging.

Precision Oncology in Lower-Grade Gliomas: Promises and Pitfalls of Therapeutic Strategies Targeting IDH-Mutations.

Simple SummaryMutations of isocitrate dehydrogenase (IDH) genes are the distinctive genetic feature of lower-grade gliomas (LGGs). Tumor-associated IDH1/2 mutations result in a loss of normal enzymatic function and the abnormal production of 2-hydroxyglutarate (2-HG), which acts as an oncometabolite causing widespread changes in histone and DNA methylation and altering cellular metabolism. In the present review, we examine the “truncal” role of IDH mutations in gliomagenesis, giving hints on the different therapeutic strategies targeting IDH1/2-mutated gliomas. We analyze in detail, preclinical and, when available, data from clinical trials of specific inhibitors blocking the mutant enzyme, IDH-targeted immunotherapeutic approaches, and agents exploiting cellular metabolic and epigenetic vulnerabilities associated with the IDH mutant phenotype.Mutations in isocitrate dehydrogenase (IDH)1 and its homolog IDH2 are considered an earliest “driver” genetic event during gliomagenesis, representing now the molecular hallmark of lower-grade gliomas (LGGs). IDH-mutated genes encode for a neomorphic enzyme that converts α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (2-HG), which accumulates to high concentrations and alters cellular epigenetics and metabolism. Targeting IDH mutations is the first attempt to apply “precision oncology” in LGGs. Two distinct strategies have been proposed so far and are under intense clinical investigation: (i) reducing the amount of intratumoral 2-HG by directly blocking the function of mutant IDH enzyme; (ii) exploiting the selective epigenetic and metabolic cellular vulnerabilities as a consequence of 2-HG accumulation. The present review describes the physiopathological mechanisms by which IDH mutations lead to tumorigenesis, discussing their prognostic significance and pivotal role in the gliomas diagnostic classification system. We critically review preclinical evidence and available clinical data of first-generation mutant-selective IDH inhibitors and novel IDH-targeted vaccines. Finally, as an alternative and attractive approach, we present the rationale to take advantage of selective 2-HG related epigenetic and metabolic weaknesses. The results of ongoing clinical trials will help us clarify the complex scenario of IDH-targeted therapeutic approaches in gliomas.

Combining Isocitrate Dehydrogenase Inhibitors With Existing Regimens in Acute Myeloid Leukemia: An Evolving Treatment Landscape.

Identification of recurrent mutations in isocitrate dehydrogenase genes (IDH1 and IDH2) in patients with acute myeloid leukemia (AML) coupled with an understanding of the pathologic role these mutant IDH isoforms impart in leukemogenesis resulted in the development of IDH1 and IDH2 inhibitors comprising a novel, molecularly defined class of targeted therapies for the treatment of AML. This review herein describes the unique cellular pathophysiology and vulnerabilities in IDH-mutated AML; the clinical development, efficacy, and known resistance mechanisms to first-generation IDH inhibitors; summarizes the literature surrounding combination therapies incorporating targeted or cytotoxic therapies with IDH inhibitors in patients with IDH-mutated AML; and identifies future challenges and areas of active ongoing investigation within this molecular subgroup.

OXA-23 β-Lactamase Overexpression in Acinetobacter baumannii Drives Physiological Changes Resulting in New Genetic Vulnerabilities.

ABSTRACTβ-Lactamase expression is the major mechanism of resistance to penicillins, cephalosporins, and carbapenems in the multidrug-resistant (MDR) bacterium Acinetobacter baumannii. In fact, stable high-level expression of at least one β-lactamase has been rapidly increasing and reported to occur in up to 98.5% of modern A. baumannii isolates recovered in the clinic. Moreover, the OXA-51 β-lactamase is universally present in the A. baumannii chromosome, suggesting it may have a cellular function beyond antibiotic resistance. However, the consequences associated with OXA β-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 β-lactamase in A. baumannii drives significant collateral changes with alterations consistent with increased amidase activity. Consequently, we predicted that these changes create new cellular vulnerabilities. As proof of principle, a small screen of random transposon insertions revealed three genes, where mutations resulted in a greater than 19-fold loss of viability when OXA-23 was overexpressed. The identified genes remained conditionally essential even when a catalytically inactive OXA-23 β-lactamase was overexpressed. In addition, we demonstrated a synergistic lethal relationship between OXA-23 overexpression and a CRISPR interference (CRISPRi) knockdown of the essential peptidoglycan synthesis enzyme MurA. Last, OXA-23 overexpression sensitized cells to two inhibitors of peptidoglycan synthesis, d-cycloserine and fosfomycin. Our results highlight the impact of OXA-23 hyperexpression on peptidoglycan integrity and reveal new genetic vulnerabilities, which may represent novel targets for antimicrobial agents specific to MDR A. baumannii and other OXA β-lactamase-overexpressing Enterobacteriaceae, while having no impact on the normal flora.

Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells

Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays.Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition.Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed “Acute Myeloid Leukemia” among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells.Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. [Display omitted] DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

Abstract 2036: Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications

Abstract Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode. Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2036.

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition.

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.

Adenylate Kinase 2 Is a Selective Dependency in NSD2-High Multiple Myeloma

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM. Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM. Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays. Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines. Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins. Disclosures No relevant conflicts of interest to declare.

LncRNA-mediated posttranslational modifications and reprogramming of energy metabolism in cancer.

Altered metabolism is a hallmark of cancer, and the reprogramming of energy metabolism has historically been considered a general phenomenon of tumors. It is well recognized that long noncoding RNAs (lncRNAs) regulate energy metabolism in cancer. However, lncRNA‐mediated posttranslational modifications and metabolic reprogramming are unclear at present. In this review, we summarized the current understanding of the interactions between the alterations in cancer‐associated energy metabolism and the lncRNA‐mediated posttranslational modifications of metabolic enzymes, transcription factors, and other proteins involved in metabolic pathways. In addition, we discuss the mechanisms through which these interactions contribute to tumor initiation and progression, and the key roles and clinical significance of functional lncRNAs. We believe that an in‐depth understanding of lncRNA‐mediated cancer metabolic reprogramming can help to identify cellular vulnerabilities that can be exploited for cancer diagnosis and therapy.

CSIG-28. ROLE OF ALTERNATIVE SPLICING IN CONFERRING SPATIAL HETEROGENEITY SIGNATURES IN GBM

Abstract Therapeutic interventions induce cell stress to trigger apoptosis in tumor cells. Specific cellular stressors, including those initiated by radio-chemotherapy, activate distinct pro-death proteins through a multitude of mechanisms. Apoptosis consists of highly regulated pathways involving post-translational modifications and cleavage of proteins leading to sequential inactivation of the main cellular processes e.g. cell cycle progression. Recently, we demonstrated that apoptotic bodies/ApoEVs in GBM transfer spliceosomal machinery components to neighboring cells, which are subsequently internalized and confer survival advantage through changes in mRNA splicing. Following the validation of the role of RBM11, a spliceosomal component in the said intercellular communication, we extended our findings in the characterization of spatially distinct clonally derived sphere lines from tumor edge and core-located tissues from GBM patients. We analyzed the expression of spliceosomal genes in edge and core-derived clones and found spatially distinct splicing capabilities and that the genes that were affected by alternative splicing events in edge and core cells have different splicing of genes that are involved in ubiquitination and regulate cell cycle progression. Downstream analyses revealed that the most promising target is MDM2. Importantly, this gene regulates cell cycle via ubiquitination. Further, western blot revealed differential expression of both RBM11 and MDM2 in egde and core-derived clones.Collectively, the data so far provides motivation for the possibility of finding cellular vulnerabilities in the splicing machinery and thus actionable targets for intervention that accounts for the bearings of spatial heterogeneity.

Abstract 2189: Evaluating efficacy of repurposed drugs in treatment of glioblastoma

Abstract Glioblastoma (GB) is the most aggressive adult brain tumor with a devastating median survival time of about fourteen months post-surgery and standard of care therapy with radiation and temozolomide. The low incidence of GB is a dis-incentive to develop novel therapies. To overcome that obstacle, we investigated the efficacy of repurposing four FDA approved drugs known to cross the BBB, minocycline, propranolol, chlorpromazine, and metformin, to inhibit signaling and metabolism in GB cells. Minocycline is a tetracycline class broad spectrum antibiotic commonly used to treat severe acne and other skin infections. Propranolol is a beta blocker type heart medication primarily used to treat high blood pressure and irregular heartbeat. Chlorpromazine is a phenothiazine antipsychotic usually used for schizophrenia. Metformin is the most widely used first-line oral treatment for type-2 diabetes. Based on a literature survey, minocycline is expected to prevent the phosphorylation of STAT3, a transcription factor downstream of EGFR; propranolol is expected to disrupt EGFR trafficking; chlorpromazine is expected to target the mTOR/AKT pathway; metformin is believed to exploit vulnerabilities in cancer cell metabolism. Efficacy of minocycline in inhibiting EGFR-driven STAT3 activation was investigated using western blot analysis. Our results demonstrate that Minocycline effectively inhibits activation of EGFR-driven STAT3 in U373 glioma cells at 100μM. The ability of chlorpromazine to inhibit the mTOR/AKT pathway was similarly tested via western blot, which showed inhibition of phosphorylated Akt and S6 at 10μM. Efficacy of propranolol in perturbing EGFR trafficking was evaluated using flow cytometry and immunofluorescence, which depicted altered membrane-associated EGFR abundance. Finally, concentration-dependent inhibition of colony formation was tested for all four drugs. Propranolol and minocycline showed potential stimulatory effects at 10μM, but all drugs inhibited cell growth at 50μM and higher. Efficacy of these drugs in treatment of GB is being further evaluated using in vitro neurosphere cultures from patients identified as having the cellular vulnerabilities potentially targeted by these drugs. Successful completion of this project will lead to in vivo efficacy testing of these four drugs in orthotopic GB PDX models. Citation Format: Tristan Neal, Nanyun Tang, George Reid, Sara Byron, Harshil Dhruv, Michael Berens. Evaluating efficacy of repurposed drugs in treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2189.