Distinct Resistance Mechanisms Research Articles

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Abstract PO3-15-03: Abemaciclib is effective in palbociclib resistant estrogen receptor positive breast cancers

Abstract Introduction: Hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (Her2)-negative breast cancer constitutes the majority of breast cancer cases and is commonly treated with endocrine therapy (ET). However, resistance to ET is a significant clinical challenge in metastatic breast cancer (MBC). The addition of cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, to ET has shown promise in delaying disease progression. Nonetheless, resistance to CDK4/6is remains a concern, necessitating the exploration of alternative treatment strategies. This study aims to investigate the distinct mechanisms of resistance between palbociclib and abemaciclib and evaluate the sequential use or targeting of differentially altered pathways to delay disease progression in HR-positive/HER2-negative tumors. Models: In vitro models of palbociclib-resistant (PR) and abemaciclib-resistant (AR) cell lines were generated to elucidate the pathways leading to resistance. Patient-derived xenografts (PDX) and ex vivo PDX-derived organoids (PDxOs) from MBC patients who experienced progression on CDK4/6is were also employed. Results: PR and AR cells exhibited unique transcriptomic and proteomic profiles, with 14 similarly and 30 differentially altered pathways identified between PR and AR in vitro models. Epithelial-mesenchymal transition (EMT) and hypoxia were enriched in PR cells but not in AR cells, while oxidative phosphorylation (OXPHOS) and reactive oxygen species pathways were enriched in AR cells. Consequently, PR cells responded to abemaciclib, whereas AR cells showed sensitivity to OXPHOS inhibitors. Genomic alterations were heterogeneous and did not consistently predict palbociclib response. Furthermore, the use of organoids and PDX models confirmed that tumors with high expression of G2/M pathway genes remained responsive to abemaciclib. Clinical data from a cohort of patients with HR-positive/HER2-negative MBC demonstrated that sequential treatment with palbociclib and abemaciclib resulted in significantly longer overall survival (42.7 months versus 17.3 months) compared to non-sequential therapy. Conclusion: Transcriptomic and proteomic analyses revealed distinct pathways in PR and AR models, suggesting that patients with enrichment of the G2/M pathway are likely to benefit from abemaciclib following palbociclib resistance, whereas those with enrichment of the OXPHOS pathway are most likely to be refractory. These findings support the need for prospective trials to evaluate the clinical benefit of abemaciclib after progression on a prior CDK4/6i regimen. Citation Format: Juliana Navarro-Yepes, Nicole Kettner, Xiayu Rao, Tuyen Bui, Cassandra Bishop, Hannah Wingate, Akshara Singareeka Raghavendra, Yan Wang, Jing Wang, Aysegul Sahin, Funda Meric-Bernstam, Kelly Hunt, Senthil Damodaran, Debu Tripathy, Khandan Keyomarsi. Abemaciclib is effective in palbociclib resistant estrogen receptor positive breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-03.

Abstract 6459: Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data

Abstract Background: Up to 85% of lung cancer patients have non-small cell lung cancer (NSCLC). Standard of care for NSCLC includes immune checkpoint inhibitors (ICI) and/or platinum-based chemotherapy (CT). However, therapy success is limited with up to 70% of patients not responding to ICI. Mechanisms of resistance across treatments are not fully understood. In this study, we explore real-world data to characterize molecular and genomic changes in advanced NSCLC treated with ICI, ICI+CT, or CT. Methods: Stage III/IV NSCLC patients receiving first-line (1L) ICI, ICI+CT, or CT were identified from Tempus Labs, Inc.’s de-identified, multimodal real-world database. Patients’ solid tissue biopsies taken pre- or post-1L underwent next generation sequencing with the Tempus xT assays (DNAseq of 648 genes at 500x coverage and whole transcriptome RNAseq). Patients’ clinical and outcome data were abstracted from electronic health records. We analyzed changes in gene expression, pathway enrichment, estimated immune cell components, and somatic mutations in responders (complete/partial response) and non-responders (progression/stable disease) upon 1L treatment. Results: 1L non-responders (70%, 51%, and 47%) were identified across cohorts (143 ICI, 764 ICI+CT, and 228 CT, respectively). 58% of pre-1L biopsies were taken from distal metastatic tissues. Distinct pathways altered post-1L include the downregulated E2F target pathway in CT cohort, and upregulated TNFα/NFκB signaling and the hypoxia and protein secretion pathways in ICI cohort. Significant (FDR < 0.05) augmented expression was observed for 90, 23, and 65 genes in non-responders vs. responders post ICI, ICI+CT, and CT, respectively. RNAseq deconvolution reveals decreased plasma and CD4+ activated T cell fraction in metastatic biopsies, whereas increased CD4+ activated T cell and monocyte fraction was seen after treatment only in ICI cohort. Genes with oncogenic driver mutations reported in NSCLC were detected including TP53 (68%), KRAS (30%), STK11 (13%), EGFR (11%), CDKN2A (10%), KEAP1 (9%), and NF1 (7%). Of the 250 mutated genes, 72 (29%) were commonly not detected post-1L, whereas 38, 3, and 8 unique genes with loss of detectable mutations were identified post-1L of ICI, ICI+CT, and CT, respectively. Since loss of detectable mutations is a sign of loss of subpopulations of cancer cells hosting such mutations that succumb to therapy, these genetic mutations could be used to select patients who can benefit from such therapy. Conclusions: Distinct molecular characteristics including tumor immune components and genomic alterations are associated with 1L response in advanced NSCLC. Real-world data analyses provide opportunity to explore distinct mechanisms of therapy resistance and identify potential biomarkers of therapy response for further validation in clinical studies. Citation Format: Xiaoyong Fu, Jordan Kardos, Paola Correa, Min Wang, Li, Jackson Egen, Shahed Iqbal. Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data [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 6459.

Abstract B058: Deconvoluting the intra-tumour heterogeneity and subclonal evolution of CDK4/6 inhibitor resistance in ER+ breast cancer

Abstract Subclonal heterogeneity and evolution are characteristics of breast cancer (BC), that play a key role in tumour development, progression, and resistance to current therapies. Preliminary single-cell sequencing studies suggest that subclonal heterogeneity drives therapy response in aggressive BC. Here, we sought to identify whether pre-existing transcriptomically defined cell subpopulations underpin resistance to CDK4/6 inhibitors (CDK4/6i) in ER+ BC, and to identify the distinct or shared resistance mechanisms between different CDK4/6i currently used in the clinic. ER+ BC cell lines, MCF7 and T47D, were infected with a lentiviral barcode library and were long-term exposed to DMSO or CDK4/6i (Abemaciclib or Palbociclib) until drug resistance was achieved. CDK4/6i resistant or DMSO cell lines were then subjected to DNA sequencing for the detection of barcodes coupled with single-cell transcriptomic and barcode sequencing, whole exome sequencing and G1/S cell cycle checkpoint analysis at a protein level. Single-cell lineage tracing identified that resistance to both CDK4/6i in MCF7 cells was stochastic, while in T47D cells, resistance to both CDK4/6i was conserved across all biological replicates. In all cell models, abemaciclib exerted a stronger selective pressure than palbociclib. Detection of barcodes from dead cells highlighted differential selective pressures of the drugs during resistance generation and upon resistance emergence. Single-cell transcriptomic sequencing identified shared and differential mechanisms of resistance between cell lines and specific CDK4/6i, suggestive of different subclonal, non-genetic mechanisms driving resistance. Some of these subclones were pre-defined (i.e. shared transcriptomic features with parental cells), whereas other resistant subclones were demarked by acquisition of pathway up-regulation. For instance, RB1 expression was abolished in the entire palbociclib-resistant T47D population, with copy number loss, indicating RB1 as a genetic driver of resistance. No evidence of RB1 loss was observed in MCF7 palbociclib-resistant clonal population, however a subclonal cluster was identified with loss of RB1 expression. Interestingly, abemaciclib-resistant MCF7 cells had lost RB1 protein expression, suggesting differential non-genetic mechanisms of resistance to CDK4/6i in MCF7 cells. Through single-cell lineage tracing, we have identified that resistance to CDK4/6i can be stochastic or conserved according to the cell model. Additionally, single-cell transcriptomic sequencing identified that in some cell models, cells that have the ability to become resistant are pre-existing, suggesting transcriptomic and epigenomic mechanisms of resistance, where cells exhibit drug-tolerant or drug-induced persistent properties. Citation Format: Ioanna Mavrommati, Fatemeh Ahmadi Moughari, Shefali Thakur, Yu Zhang, Lesley-Ann Martin, Syed Haider, Rachael Natrajan. Deconvoluting the intra-tumour heterogeneity and subclonal evolution of CDK4/6 inhibitor resistance in ER+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B058.

Alkyl deoxyglycoside-polymyxin combinations against critical priority carbapenem-resistant gram-negative bacteria

The escalating antimicrobial resistance crisis urges the development of new antibacterial treatments with innovative mechanisms of action, particularly against the critical priority carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA) and Enterobacteriaceae (CRE). Membrane-disrupting dodecyl deoxyglycosides have been reported for their interesting phosphatidylethanolamine-associated bactericidal activity against Gram-positive strains; however, their inability to penetrate the Gram-negative outer membrane (OM) renders them useless against the most challenging pathogens. Aiming to repurpose alkyl deoxyglycosides against Gram-negative bacteria, this study investigates the antimicrobial effects of five reference compounds with different deoxygenation patterns or anomeric configurations in combination with polymyxins as adjuvants for enhanced OM permeability. The generation of the lead 4,6-dideoxy scaffold was optimized through a simultaneous dideoxygenation step and applied to the synthesis of a novel alkyl 4,6-dideoxy C-glycoside 5, herein reported for the first time. When combined with subtherapeutic colistin concentrations, most glycosides demonstrated potent antimicrobial activity against several multidrug-resistant clinical isolates of CRAB, CRE and CRPA exhibiting distinct carbapenem resistance mechanisms, together with acceptable cytotoxicity against human HEK-293T and Caco-2 cells. The novel 4,6-dideoxy C-glycoside 5 emerged as the most promising prototype structure for further development (MIC 3.1 μg/mL when combined with colistin 0.5 μg/mL against CRPA or 0.25 μg/mL against several CRE and CRAB strains), highlighting the potential of C-glycosylation for an improved bioactive profile. This study is the first to show the potential of IM-targeting carbohydrate-based compounds for the treatment of infections caused by MDR Gram-negative pathogens of clinical importance.

Forward Genetic Screens Identify Mechanisms of Resistance to Small-Molecule Lactate Dehydrogenase Inhibitors.

Altered metabolism is a hallmark of cancer; however, it has been difficult to specifically target metabolism in cancer for therapeutic benefit. Cancers with genetically defined defects in metabolic enzymes constitute a subset of cancers where targeting metabolism is potentially accessible. Hürthle cell carcinoma of the thyroid (HTC) tumors frequently harbor deleterious mitochondrial DNA (mtDNA) mutations in subunits of complex I of the mitochondrial electron transport chain (ETC). Previous work has shown that HTC models with deleterious mtDNA mutations exhibit mitochondrial ETC defects that expose lactate dehydrogenase (LDH) as a therapeutic vulnerability. Here, we performed forward genetic screens to identify mechanisms of resistance to small-molecule LDH inhibitors. We identified two distinct mechanisms of resistance: upregulation of an LDH isoform and a compound-specific resistance mutation. Using these tools, we demonstrate that the anticancer activity of LDH inhibitors in cell line and xenograft models of complex I mutant HTC is through on-target LDH inhibition.

Utilizing transcriptomics and metabolomics to unravel key genes and metabolites of maize seedlings in response to drought stress

BackgroundDrought stress can substantially restrict maize growth and productivity, and global warming and an increasing frequency of extreme weather events are likely to result in more yield losses in the future. Therefore, unraveling the molecular mechanism underlying the response to drought stress is essential for breeding drought-resilient crops.ResultsIn this study, we subjected the 3-leaf-period plants of two maize inbred lines, a drought-tolerant line (si287) and a drought-sensitive line (X178), to drought stress for seven days while growing in a chamber. Subsequently, we measured physiological traits and analyzed transcriptomic and metabolic profiles of two inbred lines. Our KEGG analysis of genes and metabolites revealed significant differences in pathways related to glycolysis/gluconeogenesis, flavonoid biosynthesis, starch and sucrose metabolism, and biosynthesis of amino acids. Additionally, our joint analysis identified proline, tryptophan and phenylalanine are crucial amino acids for maize response to drought stress. Furthermore, we concentrated on tryptophan (Trp), which was found to enhance tolerance via IAA-ABA signaling, as well as SA and nicotinamide adenine dinucleotide (NAD) consequent reactive oxygen species (ROS) scavenging. We identified three hub genes in tryptophan biosynthesis, indole-3-acetaldehyde oxidase (ZmAO1, 542,228), catalase 1 (ZmCAT1, 542,369), and flavin-containing monooxygenase 6 (ZmYUC6, 103,629,142), High expression of these genes plays a significant role in regulating drought tolerance. Two metabolites related to tryptophan biosynthesis, quinolinic acid, and kynurenine improved maize tolerance to drought stress by scavenging reactive oxygen species.ConclusionsThis study illuminates the mechanisms underlying the response of maize seedlings to drought stress. Especially, it identifies novel candidate genes and metabolites, enriching our understanding of the role of tryptophan in drought stress. The identification of distinct resistance mechanisms in maize inbred lines will facilitate the exploration of maize germplasm and the breeding of drought-resilient hybrids.

A Review of Resistance to Polymyxins and Evolving Mobile Colistin Resistance Gene (mcr) among Pathogens of Clinical Significance.

The global rise in antibiotic resistance in bacteria poses a major challenge in treating infectious diseases. Polymyxins (e.g., polymyxin B and colistin) are last-resort antibiotics against resistant Gram-negative bacteria, but the effectiveness of polymyxins is decreasing due to widespread resistance among clinical isolates. The aim of this literature review was to decipher the evolving mechanisms of resistance to polymyxins among pathogens of clinical significance. We deciphered the molecular determinants of polymyxin resistance, including distinct intrinsic molecular pathways of resistance as well as evolutionary characteristics of mobile colistin resistance. Among clinical isolates, Acinetobacter stains represent a diversified evolution of resistance, with distinct molecular mechanisms of intrinsic resistance including naxD, lpxACD, and stkR gene deletion. On the other hand, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa are usually resistant via the PhoP-PhoQ and PmrA-PmrB pathways. Molecular evolutionary analysis of mcr genes was undertaken to show relative relatedness across the ten main lineages. Understanding the molecular determinants of resistance to polymyxins may help develop suitable and effective methods for detecting polymyxin resistance determinants and the development of novel antimicrobial molecules.

Antibody dependent cell-mediated cytotoxicity selection pressure induces diverse mechanisms of resistance

ABSTRACT Targeted monoclonal antibody therapy has emerged as a powerful therapeutic strategy for cancer. However, only a minority of patients have durable responses and the development of resistance remains a major clinical obstacle. Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial therapeutic mechanism of action; however, few studies have explored ADCC resistance. Using multiple in vitro models of ADCC selection pressure, we have uncovered both shared and distinct resistance mechanisms. Persistent ADCC selection pressure yielded ADCC-resistant cells that are characterized by a loss of NK cell conjugation and this shared resistance phenotype is associated with cell-line dependent modulation of cell surface proteins that contribute to immune synapse formation and NK cell function. We employed single-cell RNA sequencing and proteomic screens to interrogate molecular mechanisms of resistance. We demonstrate that ADCC resistance involves upregulation of interferon/STAT1 and DNA damage response signaling as well as activation of the immunoproteasome. Here, we identify pathways that modulate ADCC sensitivity and report strategies to enhance ADCC-mediated elimination of cancer cells. ADCC resistance could not be reversed with combinatorial treatment approaches. Hence, our findings indicate that tumor cells utilize multiple strategies to inhibit NK cell mediated-ADCC. Future research and development of NK cell-based immunotherapies must incorporate plans to address or potentially prevent the induction of resistance.

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

Distinct Mechanisms of Resistance to CDK4/6 Inhibitors Require Specific Subsequent Treatment Strategies: One Size Does Not Fit All.

Cyclin-dependent kinase (CDK) 4/6 inhibitors have transformed the treatment landscape of patients with hormone receptor-positive breast cancers. However, despite improvements in clinical outcomes, the approximately 70% of patients with tumors that are not intrinsically resistant to a CDK4/6 inhibitor still ultimately acquire resistance, which leads to a dilemma for clinicians when deciding which treatment to offer patients when they demonstrate disease progression on a CDK4/6 inhibitor. As such, many groups have sought to understand the mechanisms of resistance to CDK4/6 inhibitors, mostly focusing on genetic alterations associated with resistance. Though several recurrent mutations have been described, they are not consistent enough to guide clinical practice or generate novel rational treatment options. Two recent publications have used transcriptomic analysis to unravel distinct mechanisms driving resistance to individual CDK4/6 inhibitors and in doing so have identified biomarkers that could potentially help identify the next course of treatment for patients following disease progression.

The genetic basis of plants' battle against witchweeds: linking immune responses to distinct resistance mechanisms.

This article comments on: Mutinda S, Mobegi FM, Hale B, Dayou O, Ateka E, Wijeratne A, Wicke S, Bellis ES, Runo S. 2023. Resolving intergenotypic Striga resistance in sorghum. Journal of Experimental Botany 74, 5294–5306.

Novel STK11 differentiation phenotype classifier STK11-DPC: Immunosuppressive tumor microenvironment (TME) and response to immune checkpoint blockade (ICB) in STK11-deficient NSCLC.

2626 Background: Loss of STK11 occurs in 20-25% of NSCLC and confers poor prognosis and resistance to ICB. STK11 loss affects lineage plasticity in murine models and differentiation phenotypes in patients. However, interactions between differentiation state, TME, and clinical outcomes are not well characterized. Methods: This is a retrospective and descriptive study across eight transcriptomic datasets of 2118 non-squamous NSCLC with 314 STK11 mutations, including 628 patients randomized to docetaxel or atezolizumab in the OAK and POPLAR clinical trials. STK11-DPC applies a validated gene signature to assess functional STK11 loss, then uses a rule-based classifier to define three differentiation lineages: “Neuroendocrine,” “TTF1-Low,” and TTF1-Positive.” TME is assessed by xCell immune deconvolution, and patient outcomes assessed by Cox proportional hazards regression models. Results: STK11-DPC significantly outperforms STK11 mutations in predicting decreased MHC-1, MHC-2, interferon-stimulated genes, and immune infiltration (P < 1e-8 for each). Each of the three differentiation groups has low immune infiltration compared to STK11 WT tumors, but we observe distinctive TME phenotypes corresponding to the Neuroendocrine and TTF1-Low groups. Neuroendocrine tumors have low expression of pro-inflammatory genes in the TNF/NFKB and JAK/STAT signaling pathways (P < 1e-10), while TTF1-Low tumors show hallmarks of increased inflammation, with higher PMN-MDSC, CD8 and NK signatures, but relative exclusion of dendritic cells (P < 1e-6 for each). These differences persist after controlling for KRAS, KEAP1, and TP53 mutations. Conversely, STK11-deficient tumors in WT-like or TTF1-Positive groups have TME more similar to STK11-WT tumors and show increased immune infiltration. STK11-DPC was prognostic when applied to OAK and POPLAR, with worse OS in Neuroendocrine (HR 1.6, P = 0.006) and TTF1-Low (HR 2.4, P = 5.8e-10) groups. Survival in both arms was equally poor in these groups. In contrast, STK11-deficient tumors with WT-Like or TTF1-Positive phenotypes had better OS with atezolizumab compared to docetaxel (HR 0.5, P = 0.03). Conclusions: STK11-DPC is highly predictive of immune exclusion and NSCLC patient outcomes. It identifies STK11-deficient subsets with increased immune infiltration and improved ICB response, and two putatively immune-resistant subgroups with markedly different TME phenotypes: Neuroendocrine tumors have an anti-inflammatory phenotype, while TTF1-Low tumors exhibit chronic inflammation. Performance of STK11-DPC as a predictive biomarker warrants further validation in additional patient cohorts, while the apparent TME differences suggest distinct and non-overlapping mechanisms of ICB resistance and may guide development of precision treatment strategies.

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanismsthat do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance invivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.

Abstract F4-1: CDK4/6 inhibitor resistance: Biological mechanisms and novel approaches

Abstract CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have revolutionized the treatment of ER+ metastatic breast cancer (MBC), significantly prolonging survival. However, this combination is not curative in MBC, and resistance to CDK4/6i represents a major challenge. A diverse array of mechanisms of CDK4/6i resistance have been described, including deregulation of the G1/S cell cycle checkpoint (i.e. Rb, Cyclin E/CDK2, CDK6, INK4s), activation of growth factor signaling pathways (receptor tyrosine kinases, Ras/MAPK pathway, PI3K/AKT pathway), and contributions from the tumor microenvironment. A detailed understanding of these mechanisms is critical for overcoming CDK4/6i resistance. In this presentation, I will discuss recent advances in defining novel biomarkers that are causally associated with CDK4/6i resistance, therapeutic vulnerabilities linked to distinct mechanisms of resistance, and potential strategies to improve clinical responses to CDK4/6i in ER+ MBC. Citation Format: Ariella Hanker. CDK4/6 inhibitor resistance: Biological mechanisms and novel approaches [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr F4-1.

RNA-Seq-Pop: Exploiting the sequence in RNA sequencing-A Snakemake workflow reveals patterns of insecticide resistance in the malaria vector Anopheles gambiae.

We provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides end-to-end analysis of RNA sequencing data sets. The workflow allows the user to perform quality control, perform differential expression analyses and call genomic variants. Additional options include the calculation of allele frequencies of variants of interest, summaries of genetic variation and population structure, and genome-wide selection scans, together with clear visualizations. RNA-Seq-Pop is applicable to any organism, and we demonstrate the utility of the workflow by investigating pyrethroid resistance in selected strains of the major malaria mosquito, Anopheles gambiae. The workflow provides additional modules specifically for An.gambiae, including estimating recent ancestry and determining the karyotype of common chromosomal inversions. The Busia laboratory colony used for selections was collected in Busia, Uganda, in November 2018. We performed a comparative analysis of three groups: a parental G24 Busia strain; its deltamethrin-selected G28 offspring; and the susceptible reference strain Kisumu. Measures of genetic diversity reveal patterns consistent with that of laboratory colonization and selection, with the parental Busia strain exhibiting the highest nucleotide diversity, followed by the selected Busia offspring, and finally, Kisumu. Differential expression and variant analyses reveal that the selected Busia colony exhibits a number of distinct mechanisms of pyrethroid resistance, including the Vgsc-995S target-site mutation, upregulation of SAP genes, P450s and a cluster of carboxylesterases. During deltamethrin selections, the 2La chromosomal inversion rose in frequency (from 33% to 86%), supporting a previous link with pyrethroid resistance. RNA-Seq-Pop is hosted at: github.com/sanjaynagi/rna-seq-pop. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An.gambiae and other taxa.

LY6D marks pre-existing resistant basosquamous tumor subpopulations

Improved response to canonical therapies requires a mechanistic understanding of dynamic tumor heterogeneity by identifying discrete cellular populations with enhanced cellular plasticity. We have previously demonstrated distinct resistance mechanisms in skin basal cell carcinomas, but a comprehensive understanding of the cellular states and markers associated with these populations remains poorly understood. Here we identify a pre-existing resistant cellular population in naive basal cell carcinoma tumors marked by the surface marker LY6D. LY6D+ tumor cells are spatially localized and possess basal cell carcinoma and squamous cell carcinoma-like features. Using computational tools, organoids, and spatial tools, we show that LY6D+ basosquamous cells represent a persister population lying on a central node along the skin lineage-associated spectrum of epithelial states with local environmental and applied therapies determining the kinetics of accumulation. Surprisingly, LY6D+ basosquamous populations exist in many epithelial tumors, such as pancreatic adenocarcinomas, which have poor outcomes. Overall, our results identify the resistant LY6D+ basosquamous population as an important clinical target and suggest strategies for future therapeutic approaches to target them.

Retreatment with Polatuzumab Vedotin in Combination with Rituximab Increases Antitumor Activity Against Polatuzumab Vedotin-Resistant DLBCL Cells

Introduction. Polatuzumab vedotin (Pola) is a monoclonal antibody against B-cell antigen CD79b conjugated to monomethyl auristatin E (MMAE), and is approved in combination with bendamustine and rituximab (Rit) in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) in various countries. Moreover, in the POLARIX study, Pola-R-CHP (cyclophosphamide, doxorubicin, and prednisone) achieved a significantly improved progression-free survival compared to R-CHOP (CHP plus vincristine) in previously untreated DLBCL. The treatment landscape of DLBCL have continued to change dynamically in recent years with the development of various agents. Although the detailed mechanism of resistance to Pola is not clear, understanding it is important for the development of next-line treatment to follow Pola-containing therapy, including retreatment with Pola. To address this, we established the Pola-resistant cells using Pola-sensitive human DLBCL and assessed the combination efficacy of Pola with Rit, the key component in the treatment of DLBCL to determine the efficacy of retreatment with Pola. Methods. We established Pola-resistant DLBCL cells from STR-428 and SU-DHL-4 cells through long-term exposure to Pola. The cell cytotoxicity to Pola was assessed by WST-8 assay. The in vitro combination efficacy of Pola plus Rit was assessed by measuring the sensitivity to complement-dependent cytotoxicity (CDC), rituximab's potent mechanism of action. In CDC assays, calcein-AM stained tumor cells were incubated with or without Rit in the medium containing 15% normal human serum. After incubating for 4 hours at 37℃, the fluorescence intensity was measured. Expression levels of protein were determined by flow cytometry or immunoblotting. For in vivo experiments, Pola-resistant STR-428 cells were injected subcutaneously into C.B-17/Icr-scid/scidJcl mice. Human IgG, Pola, or Rit was administered intravenously on Day 1. Results. Pola-resistant cells established from SU-DHL-4 cells (SU-DHL-4-Pola-R) showed a 2.3-fold higher IC50 value than parental cells. In SU-DHL-4-Pola-R cells, there was an increase in the expression of MDR1 (1.8-fold), a multidrug resistance pump mediating intracellular MMAE efflux. Interestingly, treatment with 1 μg/mL of Pola for 7 days further increased the IC50 value (16.8-fold) and the expression of MDR1 (3.6-fold）in SU-DHL-4-Pola-R cells.MDR1 inhibitor verapamil increased sensitivity to Pola, indicating that overexpression of MDR1 is one of the mechanisms of resistance to Pola in these cells. We then investigated the efficacy of combination therapy with Rit against Pola-resistant cells. Pretreatment with Pola or anti-CD79b antibody before the assays enhanced Rit-induced amount of membrane attack complex during complement activation and significantly increased CDC sensitivity in SU-DHL-4-Pola-R cells. Other Pola-resistant cells derived from STR-428 cells (STR-428-Pola-R) displayed an IC50 value 17.6-fold higher than the parental cells. STR-428-Pola-R cells did not exhibit Pola-resistant factors such as MDR1 upregulation or loss of CD79b expression, but showed decreased expression of Bim, an apoptosis-inducing factor. Stable knockdown of Bim reduced the sensitivity to Pola in parental STR-428 cells, indicating decreased Bim expression could be the resistant mechanism to Pola in these cells. In STR-428-Pola-R cells, 3 days pretreatment with Pola or MMAE significantly increased Rit-induced CDC sensitivity. Immunoblotting analysis revealed that pretreatment with Pola or MMAE downregulated the expression of Mcl-1, which is known to attenuate CDC sensitivity. Mcl-1 inhibitor AZD5991 significantly increased CDC sensitivity, suggesting that Pola-mediated downregulation of Mcl-1 could upregulate the sensitivity to CDC in these cells. In addition, treatment with Pola (1 mg/kg) plus Rit (10 mg/kg) significantly enhanced antitumor activity in mouse xenograft models derived from STR-428-Pola-R cells. Conclusion. Pretreatment with Pola enhanced Rit-induced CDC sensitivity in two Pola-resistant cells with distinct mechanisms of resistance: increased MDR1 or decreased Bim expression. Combination treatment of Pola with Rit enhanced antitumor activity in STR-428-Pola-R xenografted mice. These results indicate that retreatment with Pola in combination with Rit could be a therapeutic option in patients with Pola-resistant tumors.

Distinct roles of treatment schemes and BRCA2 on the restoration of homologous recombination DNA repair and PARP inhibitor resistance in ovarian cancer.

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a major advance in ovarian cancer, now as a treatment and as a maintenance therapy in the upfront and recurrent settings. However, patients often develop resistance to PARPis, underlining the importance of dissecting resistance mechanisms. Here, we report different dosing/timing schemes of PARPi treatment in BRCA2-mutant PEO1 cells, resulting in the simultaneous development of distinct resistance mechanisms. PARPi-resistant variants PEO1/OlaJR, established by higher initial doses and short-term PARPi treatment, develops PARPi resistance by rapidly restoring functional BRCA2 and promoting drug efflux activity. In contrast, PEO1/OlaR, developed by lower initial doses with long-term PARPi exposure, shows no regained BRCA2 function but a mesenchymal-like phenotype with greater invasion ability, and exhibits activated ATR/CHK1 and suppressed EZH2/MUS81 signaling cascades to regain HR repair and fork stabilization, respectively. Our study suggests that PARPi resistance mechanisms can be governed by treatment strategies and have a molecular basis on BRCA2 functionality. Further, we define different mechanisms that may serve as useful biomarkers to assess subsequent treatment strategies in PARPi-resistant ovarian cancer.

The distinct resistance mechanisms of cyanobacteria and green algae to sulfamethoxazole and its implications for environmental risk assessment

Cyanobacteria and green algae are the OECD recommended test organisms for environmental toxicity assessments of chemicals. Whether the differences in these two species' responses to the identical chemical affect the assessment outcomes is a question worth investigating. Firstly, we investigated the distinct resistance mechanisms of Synechococcus sp. (cyanobacteria) and R. subcapitata (green algae) to sulfamethoxazole (SMX). The antioxidant system analysis demonstrated that R. subcapitata mainly relies on enhancing the activity of first line defense antioxidants, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which is the most powerful and efficient response to get rid of ROS, whereas Synechococcus sp. depends upon increasing the activity of glutathione-S-transferase (GST) and GPx to resist oxidative stress. Besides, a total 7 transformation products (TPs) of SMX were identified in R. subcapitata culture medium. The analysis of conjectural transformation pathways and the predicted toxicity indicates that R. subcapitata could relieve SMX toxicity by degrading it to low eco-toxic TPs. Additionally, we summarized numerous exposure data and assessed the environmental risk of various antibiotics, revealing an inconsistent result for the same type of antibiotic by using cyanobacteria and green algae, which is most likely due to the different resistance mechanisms. In the future, modified indicators or comprehensive assessment methods should be considered to improve the rationality of environmental toxicity assessments.