Multiple Resistance Mechanisms Research Articles

Characterization and fitness cost of bifenthrin resistance in Rhopalosiphum padi (Hemiptera: Aphididae).

Rhopalosiphum padi is an important global wheat pest. The pyrethroid insecticide bifenthrin is widely used in the control R. padi. We explored the resistance potential, cross-resistance, adaptive costs, and resistance mechanism of R. padi to bifenthrin using a bifenthrin-resistant strain (Rp-BIF) established in laboratory. The Rp-BIF strain developed extremely high resistance against bifenthrin (1033.036-fold). Cross-resistance analyses showed that the Rp-BIF strain had an extremely high level of cross-resistance to deltamethrin (974.483-fold), moderate levels of cross-resistance to chlorfenapyr (34.051-fold), isoprocarb (27.415-fold), imidacloprid (14.819-fold), and thiamethoxam (11.228-fold), whereas negative cross-resistance was observed to chlorpyrifos (0.379-fold). The enzymatic activity results suggested that P450 played an important role in bifenthrin resistance. A super-kdr mutation (M918L) of voltage-gated sodium channel (VGSC) was found in the bifenthrin-resistant individuals. When compared with the susceptible strain (Rp-SS), the Rp-BIF strain was significantly inferior in multiple life table parameters, exhibiting a relative fitness of 0.69. Our toxicological and biochemical studies indicated that multiple mechanisms of resistance might be involved in the resistance trait. Our results provide insight into the bifenthrin resistance of R. padi and can contribute to improve management of bifenthrin-resistant R. padi in the field.

Multiple mechanisms of linezolid resistance in Staphylococcus haemolyticus detected by whole-genome sequencing.

Introduction. Linezolid is an effective therapeutic option for treating severe infections caused by multidrug-resistant Gram-positive organisms. Several mechanisms have been reported to be responsible for resistance to this antibiotic.Hypothesis or Gap Statement. Although several mechanisms of linezolid resistance have been reported in Staphylococcus haemolyticus, the prevalence and potential for horizontal transfer of resistance genes have not been fully characterized, particularly among S. haemolyticus isolates from India.Aim. To perform whole-genome sequencing (WGS) of linezolid-resistant S. haemolyticus isolates to characterize the resistance mechanisms.Methodology. WGS was performed for 16 linezolid-resistant S. haemolyticus isolates to check for the presence of cfr, optrA and poxtA genes and mutations in 23S rRNA and ribosomal proteins (L3, L4 and L22) that are possible mechanisms implicated in linezolid resistance. Sequence types were identified using MLST finder. The minimum inhibitory concentration (MIC) of linezolid was determined using the E-test method. Polymerase chain reaction (PCR) was carried out for the detection of the cfr gene.Results. The study documented three different mechanisms of linezolid resistance in S. haemolyticus. Thirteen of the 16 isolates were phenotypically resistant to linezolid, of which 12 were positive for the cfr gene. The G2603T mutation in 23S rRNA was found in the majority of the isolates (n=13). Ten isolates had the R138V mutation in L3 ribosomal protein. Twelve isolates with the cfr gene in combination with either G2603T or R138V mutations displayed extremely high MIC values. Surprisingly, three phenotypically sensitive isolates were found to be positive for the cfr gene but negative for other resistance mechanisms. Importantly, in almost half of the isolates the cfr gene was present on a plasmid. ST3 and ST1 were found to be the predominant sequence types.Conclusion. All phenotypically resistant isolates exhibited two or three linezolid resistance mechanisms. The cfr gene was found on plasmids in many isolates, demonstrating its potential for horizontal transfer to more pathogenic organisms.

Emergence of ST463 exoU-Positive, Imipenem-Nonsusceptible Pseudomonas aeruginosa Isolates in China.

This study investigated the resistance mechanisms and the distribution and proportions of virulence genes, including exoU, in 182 imipenem-nonsusceptible Pseudomonas aeruginosa (INS-PA) strains collected from China in 2019. There was no obvious prevalent sequence type or concentrated evolutionary multilocus sequence typing (MLST) type on the INS-PA phylogenetic tree in China. All of the INS-PA isolates harbored β-lactamases with/without other antimicrobial mechanisms, such as gross disruption of oprD and overexpression of efflux genes. Compared with exoU-negative isolates, exoU-positive isolates (25.3%, 46/182) presented higher virulence in A549 cell cytotoxicity assays. The southeast region of China had the highest proportion (52.2%, 24/46) of exoU-positive strains. The most frequent exoU-positive strains belonged to sequence type 463 (ST463) (23.9%, 11/46) and presented multiple resistance mechanisms and higher virulence in the Galleria mellonella infection model. The complex resistance mechanisms in INS-PA and the emergence of ST463 exoU-positive, multidrug-resistant P. aeruginosa strains in southeast China indicated a challenge that might lead to clinical treatment failure and higher mortality. IMPORTANCE This study investigates the resistance mechanisms and distribution and proportions of virulence genes of imipenem-nonsusceptible Pseudomonas aeruginosa (INS-PA) isolates in China in 2019. Harboring PDC and OXA-50-like genes is discovered as the most prevalent resistance mechanism in INS-PA, and the virulence of exoU-positive INS-PA isolates was significantly higher than that of exoU-negative INS-PA isolates. There was an emergence of ST463 exoU-positive INS-PA isolates in Zhejiang, China, most of which presented multidrug resistance and hypervirulence.

Psychrobacter Phage Encoding an Antibiotics Resistance Gene Represents a Novel Caudoviral Family.

Psychrobacter is an important bacterial genus that is widespread in Antarctic and marine environments. However, to date, only two complete Psychrobacter phage sequences have been deposited in the NCBI database. Here, the novel Psychrobacter phage vB_PmaS_Y8A, infecting Psychrobacter HM08A, was isolated from sewage in the Qingdao area, China. The morphology of vB_PmaS_Y8A was characterized by transmission electron microscopy, revealing an icosahedral head and long tail. The genomic sequence of vB_PmaS_Y8A is linear, double-stranded DNA with a length of 40,226 bp and 44.1% G+C content, and encodes 69 putative open reading frames. Two auxiliary metabolic genes (AMGs) were identified, encoding phosphoadenosine phosphosulfate reductase and MarR protein. The first AMG uses thioredoxin as an electron donor for the reduction of phosphoadenosine phosphosulfate to phosphoadenosine phosphate. MarR regulates multiple antibiotic resistance mechanisms in Escherichia coli and is rarely found in viruses. No tRNA genes were identified and no lysogeny-related feature genes were detected. However, many similar open reading frames (ORFs) were found in the host genome, which may indicate that Y8A also has a lysogenic stage. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis indicate that vB_PmaS_Y8A contains a novel genomic architecture similar only to that of Psychrobacter phage pOW20-A, although at a low similarity. vB_PmaS_Y8A represents a new family-level virus cluster with 22 metagenomic assembled viral genomes, here named Minviridae. IMPORTANCE Although Psychrobacter is a well-known and important bacterial genus that is widespread in Antarctic and marine environments, genetic characterization of its phages is still rare. This study describes a novel Psychrobacter phage containing an uncharacterized antibiotic resistance gene and representing a new virus family, Minviridae. The characterization provided here will bolster current understanding of genomes, diversity, evolution, and phage-host interactions in Psychrobacter populations.

Managing pesticide resistance in Spodoptera frugiperda: A spatially explicit framework for identifying optimal treatment strategies

Pesticide resistance is a major challenge to increasing the resilience and sustainability of current food production systems. Preserving the susceptibility of pest organisms to chemical products is a key factor to optimize a pesticide-based strategy. However, resistance management strategies (RMSs) must consider unique species biologies, multiple resistance mechanisms, environmental factors, and pest management practices, which can make their implementation complex.Here, we develop a method to help manage this complexity using a grid-based simulation framework for pesticide resistance evolution including population growth and dispersal dynamics. This framework was applied to the fall armyworm, Spodoptera frugiperda, for which resistance evolution is a major concern. We explored the sensitivity of 13 parameters dealing with landscape structure, dispersal rate, chemical treatment protocols, chemical degradation rate, dose-response curves and transition rates (i.e., flux between sub-population driven by the mutation rate). From the sensitivity analysis of simulations, we computed heat maps of the influence of each parameter on a set of variables (total pest population size, fully resistant population size, and resistance frequency).Assuming a large but realistic range for each parameter, Sobol's sensitivity index showed that resistant transition rate (from phenotypically susceptible to resistant sub-populations) and pesticide properties (in particular, degradation rate and dose-response curve) are more important in the outbreak of resistance compared with resistance ratio (i.e., the benefit of being resistant over susceptible in terms of fitness), chemical application intensity and landscape composition. In addition, using Pareto optimality, we assessed the performance of different pesticide application regimes according to total population size, population size of resistant individuals, the total amount of pesticide, and the total area of host plants suitable for S. frugiperda. Across the wide parameter space explored, we revealed the high volatility of outputs suggesting that the performance of treatment protocols depends on the ecological context. Nevertheless, despite this variability, a “windowing” management strategy consisting of a single pesticide group applied per insect generation, provided the optimal control of S. frugiperda and resistance evolution outcomes.This work provides a set of tools to test RMS scenarios for the control of S. frugiperda and to understand how variabilities may arise at different management steps and geographical scales.

PIK3CB as a potential target to regulate chemosensitivity in glioblastoma.

e14051 Background: Glioblastoma (GBM) is the most common primary central nervous system malignancy but exhibits universally poor outcomes in part due to chemoresistance. Temozolomide (TMZ) can significantly improve overall survival, but resistance mechanisms limit its consistent efficacy. The phosphoinositide 3 kinase (PI3K) pathway is involved in multiple TMZ resistance mechanisms and is highly druggable; however, pan-inhibition of its four grossly homologous but functionally distinct catalytic subunits (PIK3CA/B/D/G encode p110α/β/δ/γ, respectively) was unsuccessful clinically. Therefore, by understanding how the individual PI3K subunits regulate TMZ sensitivity, a more selective treatment strategy can be developed to combat chemoresistance. Methods: Differential expression of PI3K subunit genes was assessed using RNA sequencing (RNAseq) results obtained from public databases (e.g., DepMap, The Cancer Tissue Genome Atlas) for GBM cell lines and patient tumors expressing high or low O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair enzyme highly implicated in TMZ resistance. PI3K protein levels were assessed using immunohistochemistry of gliomas available at The Human Protein Atlas (THPA). PI3K gene expression was correlated with PI3K pathway activation, quantified as protein kinase B phosphorylation (pAKT). PI3K mRNAs and pAKT were then correlated with the half maximal inhibitory concentrations (IC50s) of TMZ in GBM cell lines to evaluate for a relationship between PI3K gene expression, PI3K pathway activity, and TMZ sensitivity. Finally, hazard ratios were used to compare PI3K gene expression in patient tumors with survival and response to chemotherapy to determine if expression was predictive of prognosis and/or chemosensitivity. Results: PIK3CB was the only PI3K gene enriched in both GBM cell lines and tumors, demonstrating significantly higher expression than all other PI3K genes in GBM (P < 0.0001), independent of MGMT levels. PIK3CB expression remained the highest even after controlling for GBM risk factors such as tumor recurrence, isocitrate dehydrogenase (IDH) mutations, molecular subtypes, or patient gender/age differences. Levels of the p110β protein were also significantly higher than those of other PI3K catalytic subunits (P < 0.001). PIK3CB expression was positively correlated with pAKT levels (P < 0.05), but only in cell lines and tumors with low MGMT activity. Both PIK3CB expression and pAKT levels correlated positively with TMZ IC50s (P < 0.05) in MGMT-deficient GBMs, suggesting an inverse relationship between PIK3CB and TMZ sensitivity. Finally, better prognosis and significant survival benefit with chemotherapy were both associated with lower levels of PIK3CB in MGMT-deficient patients. Conclusions: PIK3CB may selectively regulate chemoresistance in GBM, suggesting a role for p110β selective inhibition to improve chemosensitivity.

Understanding and targeting resistance mechanisms in cancer.

Resistance to cancer therapies has been a commonly observed phenomenon in clinical practice, which is one of the major causes of treatment failure and poor patient survival. The reduced responsiveness of cancer cells is a multifaceted phenomenon that can arise from genetic, epigenetic, and microenvironmental factors. Various mechanisms have been discovered and extensively studied, including drug inactivation, reduced intracellular drug accumulation by reduced uptake or increased efflux, drug target alteration, activation of compensatory pathways for cell survival, regulation of DNA repair and cell death, tumor plasticity, and the regulation from tumor microenvironments (TMEs). To overcome cancer resistance, a variety of strategies have been proposed, which are designed to enhance the effectiveness of cancer treatment or reduce drug resistance. These include identifying biomarkers that can predict drug response and resistance, identifying new targets, developing new targeted drugs, combination therapies targeting multiple signaling pathways, and modulating the TME. The present article focuses on the different mechanisms of drug resistance in cancer and the corresponding tackling approaches with recent updates. Perspectives on polytherapy targeting multiple resistance mechanisms, novel nanoparticle delivery systems, and advanced drug design tools for overcoming resistance are also reviewed.

Abstract B004: Multi-omics profiling to identify mechanisms of resistance to KRAS inhibition: A comparative study on colorectal and lung cancer

Abstract Background: Lung adenocarcinoma (LUAD) has a higher objective response rate to single-agent KRASG12C inhibitors (e.g., AMG 510) than colorectal cancer (CRC) patients (32.2% vs 7.1%). It is known that molecular heterogeneities exist between KRASMut LUAD and CRC tissues. We hypothesize that co-mutations and oncogenic pathways may confer resistance to KRAS inhibitors in CRC cells. Methods: Whole-exome and transcriptome profiles of KRASmut LUAD (n=168) and microsatellite stable (MSS) CRC (n=183) patients were downloaded from TCGA. Mutation profiles of KRASmut patients were also obtained from AACR Project-GENIE. Gene essentiality scores derived from the whole genome CRISPR screenings were downloaded from the DepMap to evaluate the relative oncogene addiction to KRAS in lung (n=133) and CRC (n=59) cell lines. Differential gene expressions studies were performed using EBayes, followed by gene set enrichment analysis (GSEA). CRC cell lines SW837, SW1463, SW403, and HCT15 were treated with AMG 510, a KRASG12C inhibitor, and Pictilisib, a PIK3CA inhibitor, and cell viability was measured up to 96 hours after treatment. Results: The KRASmut LUAD cohort had co-mutation clusters remarkable for (i) no co-mutation (59%, 99/168), (ii) KEAP1mut and/or STK11mut (20.2%, 34/168), and (iii) a mixed group with a frequent mutation in other genes (mean=2) but without a clear recurrent pattern (20.8%, 35/168). In CRC, KRASmut tumors formed clusters of (i) no co-mutation (59.6%, 109/183), (ii) PIK3CAmut (26.2%, 48/183), and (iii) a similar cluster with frequent co-mutation (mean=2) without a recurrent pattern (14.2%, 26/183). Similar co-mutation patterns were also observed in AACR Project-GENIE LUAD and CRC cohorts. KRAS oncogene addiction was seen in both KRASMut lung (median scores, -1.1 vs -0.4, p<0.001) and CRC (median scores, -1.3 vs -0.5, p<0.001) cell lines than KRASwt cells. However, KRASmut/PIK3CAmut co-mutated CRC cell lines (n=10) were significantly less dependent on the KRAS gene than KRASmut/PIK3CAwt cells (n=22) (median scores, -1.0 vs -1.4, p=0.03). Two KRASG12C/PIK3CAwt CRC cell lines that are highly dependent on KRAS (SW837 and SW1463 cells) also showed higher sensitivities (IC50<5uM) to AMG 510 and Pictilisib relative to KRASnon-G12C/PIK3CAmut CRC cell lines SW403 and HCT15 (IC50>10uM). GSEA revealed that the Hedgehog, spermatogenesis, and pancreas-beta cells genesets were significantly active (Normalized enrichment scores > 1, FDR<0.05) in KRAS KO-resistant CRC cells, but the same genesets were not enriched in KRAS KO-resistant lung cancer cells. Conclusion: Somatic co-mutations and transcriptomics signatures in KRASmut LUAD and CRC may predict sensitivities to KRAS inhibitors. KRASmut CRC cells with PIK3CAmut or active hedgehog pathway are resistant to KRAS gene KO, suggesting multiple drug resistance mechanisms to KRAS inhibitors exist in KRASmut CRC cells. Future studies should emphasize identifying new drug targets, as optimal drug combinations of KRAS inhibitors may not be same for all KRASmut CRC patients. Citation Format: Saikat Chowdhury, Vinay K. Pattalachinti, Valsala Haridas, Scott Kopetz, John Paul Shen. Multi-omics profiling to identify mechanisms of resistance to KRAS inhibition: A comparative study on colorectal and lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B004.

PARP Inhibitors in Breast and Ovarian Cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors are one of the most successful examples of clinical translation of targeted therapies in medical oncology, and this has been demonstrated by their effective management of BRCA1/BRCA2 mutant cancers, most notably in breast and ovarian cancers. PARP inhibitors target DNA repair pathways that BRCA1/2-mutant tumours are dependent upon. Inhibition of the key components of these pathways leads to DNA damage triggering subsequent critical levels of genomic instability, mitotic catastrophe and cell death. This ultimately results in a synthetic lethal relationship between BRCA1/2 and PARP, which underpins the effectiveness of PARP inhibitors. Despite the early and dramatic response seen with PARP inhibitors, patients receiving them often develop treatment resistance. To date, data from both clinical and preclinical studies have highlighted multiple resistance mechanisms to PARP inhibitors, and only by understanding these mechanisms are we able to overcome the challenges. The focus of this review is to summarise the underlying mechanisms underpinning treatment resistance to PARP inhibitors and to aid both clinicians and scientists to develop better clinically applicable assays to better select patients who would derive the greatest benefit as well as develop new novel/combination treatment strategies to overcome these mechanisms of resistance. With a better understanding of PARP inhibitor resistance mechanisms, we would not only be able to identify a subset of patients who are unlikely to benefit from therapy but also to sequence our treatment paradigm to avoid and overcome these resistance mechanisms.

Abstract LB320: Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor

Abstract The RAS family of proto-oncogenes are the most frequently mutated genes in cancer, in which mutations in KRAS account for approximately 25% of all human cancers. RAS oncogenes impair the ability of RAS to convert from its active GTP-bound form into its inactive GDP-bound state leading to the sustained activation of the MAPK signaling pathway and ultimately driving tumorigenesis. Until recently, KRAS was regarded as undruggable, but newly approved covalent inhibitors of KRASG12C have demonstrated clinical benefit in patients harboring this mutation. However, patients with KRASG12C mutation only represent approximately 10% of the KRAS-mutated or amplified cancer population, and multiple RAS-mediated resistance mechanisms have emerged with these covalent compounds. Here we show that QTX3034 is a potent, highly selective, and orally bioavailable mutant KRAS inhibitor. Using the most common KRAS mutant, KRASG12D, QTX3034 demonstrated picomolar binding affinity (0.6 nM) to the inactive form of KRASG12D and inhibited SOS1/2 mediated nucleotide exchange with picomolar potency (0.2 nM). In a NanoBiT cellular target engagement assay, QTX3034 treatment disrupted KRASG12D:RAF1 protein-protein interaction in a concentration-dependent manner with an EC50 of 17 nM in HEK293T cells. Western blot analyses and CTG proliferation assays illustrated that QTX3034 treatment inhibited downstream ERK phosphorylation and cell proliferation in KRASG12D -driven cancer cell lines. Similar inhibitory effects were observed across KRASG12D and KRASG12V mutant cancer cell lines, albeit less potent against the latter. Notably, QTX3034 produced no effect against NRAS or BRAF mutant cells. In vivo, QTX3034 achieved sustained systemic exposure levels required for efficacy following single oral administration in mice, indicating potential durable inhibition of KRAS signaling in vivo. In a KRASG12D -driven xenograft model, oral administration of QTX3034 twice daily (BID) demonstrated dose-dependent anti-tumor efficacy and achieved tumor regression in 100% of tumors. In a KRASG12V -driven xenograft model, oral administration of QTX3034 showed significant tumor growth inhibition, consistent with its activity against KRASG12V in vitro. QTX3034 was well-tolerated in rodent, repeat dose toxicology studies and exhibited significant exposure in the brain. QTX3034 has good solubility, permeability, and moderate protein binding in human plasma. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3034 exhibited weak CYP inhibition (direct and time-dependent) with a favorable in vitro off-target selectivity/safety profile. Taken together, this preclinical characterization supports the advancement of QTX3034 into IND-enabling studies for KRAS mutant cancers. Citation Format: Yang W. Zhang, Dave Rominger, Elizabeth Donohue Vo, Jillian M. Silva, Yang J. Zhang, Greg Lee, John Micozzi, Ben Reid, Brooke McDonough, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB320.

Abstract CT188: A phase Ib study of sapacitabine (sapa) and olaparib (ola) in patients (pts) with BRCA1/2-mutated (BRCA1/2m) metastatic breast cancer (MBC)

Abstract Background: PARP inhibitors (PARPi) are superior to chemotherapy in pts with BRCA1/2m MBC, with median PFS (mPFS) of 7 months with single-agent ola. Sapa is an oral nucleoside analog; the active metabolite, CNDAC, generates single-strand DNA breaks that are converted to double-strand breaks (DSB) during subsequent replication, resulting in cell death. CNDAC-induced DSB repair is dependent on homologous recombination. Sapa combined with ola may augment DNA damage compared to ola alone in BRCA1/2m tumors. Methods: We conducted a phase Ib study of sapa/ola in pts with BRCA1/2m HER2-negative MBC who were not previously PARPi-exposed. Primary objective was to determine the RP2D of sapa with ola. Secondary objectives included safety, tolerability and mPFS. RAD51 staining was performed on archival tissue. Serial blood samples were collected for circulating tumor DNA (ctDNA) analysis using low-pass WGS with targeted sequencing via the DDR Discovery Panel (Resolution Bioscience). Results: 10 women (median age 46) were enrolled before the study was terminated for hematologic toxicity. Germline BRCA1 and BRCA2 mutations were present in 3 and 7 pts, respectively. 4 pts had received prior platinum. All pts initially received standard dose ola (300 mg twice daily) with 100 mg (n = 3), 150 mg (n = 4), or 200 mg (n = 3) sapa once daily on days 1-5 and 8-12 of an every 28-day cycle. The RP2D was not determined. Hematologic AEs were common, including anemia (80%), neutropenia (80%), and thrombocytopenia (40%), with 1 DLT of G4 neutropenia lasting more than 7 days. The ORR was 50% (95% CI: 18.7% - 81.3%) with mPFS of 9.7 months (95% CI: 8.02 - NA). 3 pts had clinical benefit of 15+ months, exceeding expectations with olaparib alone, including 2 with PR, and 2 who remained on trial for 36+ months. 3 patients had confirmed PRs of short duration. Of the remaining patients, one withdrew consent early and the other 3 had non-CR/non-PD or SD of 4, 7 and 9 months. At progression, 2 pts had evidence of BRCA reversion mutation associated with an MMEJ signature; ctDNA also demonstrated MRE11 and PTEN amplifications in 1 of these pts, suggesting multiple resistance mechanisms. 3 pts had no evidence of reversion but had acquired putative non-reversion mechanisms of resistance, including changes suggesting enhanced MRN complex function, FANCD2 and ATR amplifications and PIK3CA mutation. The majority of tumors (6/10) were RAD51-negative. Other tumors with RAD51 foci demonstrated replication stress evidenced by high levels of pKAP1 and pRPA, suggesting benefit associated with sapa. Conclusions: Sapa/ola produces high rates of hematologic toxicity. However, the ORR of 50%, mPFS of 9.7 months, and durability of 3+ years in 2 pts suggest possible combinatorial benefit. Further exploration with different sapa schedules or with substitution of a PARP1-selective inhibitor to decrease hematologic toxicity is warranted. Citation Format: Filipa Lynce, Noah Graham, Bose S. Kochupurakkal, Huy Nguyen, Philip D. Poorvu, Victoria Attaya, Raechel Davis, Kavita Garg, Lee P. Lim, Mark Li, Cloud P. Paweletz, Nabihah Tayob, Geoffrey I. Shapiro, Sara M. Tolaney. A phase Ib study of sapacitabine (sapa) and olaparib (ola) in patients (pts) with BRCA1/2-mutated (BRCA1/2m) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT188.

Abstract 3907: Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor

Abstract Resistance to targeted therapies is a major challenge in oncology. Disease progression is caused by multiple resistance mechanisms. Besides pre-existing and acquired genetic alternations, adaptive non-mutational reprogramming as well as modulation of phenotypic plasticity have emerged as drivers of disease progression. For example, in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) patients who were treated with osimertinib, ~50% of progressive disease could not be attributed to genetic mutations (Leonetti et al., BJC 2019). Similarly, in ~ 40% of patients suffering from KRAS G12C-mutated NSCLC and colorectal cancer (CRC), disease progressed under sotorasib treatment without identifiable acquired mutations (Zhao et al., Nature 2021). Thus, targeting non-genetic adaptive resistance mechanism such as drug-induced transcriptional reprogramming might be of great therapeutic benefit. Here we identified small molecules, that interfere with cancer drug-induced transcriptional escape mechanisms using a phenotypic screen based on an SRY-Box Transcription Factor 2 (SOX2) reporter system. The screen led to the development of TT125-802, a highly specific and potent, orally available small molecule inhibitor of the bromodomain of the transcriptional and epigenetic regulator CBP [cyclic adenosine monophosphate response element binding protein (CREB) binding protein] and its paralogue p300. TT125-802 dose-dependently prevented osimertinib resistance development in EGFR-mutated NSCLC cell lines HCC827 and HCC4006, as well as sotorasib resistance development in KRAS G12C-mutated NCI-H358 (NSCLC), SW837 and SNU-1411 (CRCs) as assessed by label-free long-term live microscopy assays. Data generated in mouse xenograft studies confirmed the ability of TT125-802 increasing response rates and prolonging the duration of response to osimertinib and sotorasib in vivo. In cells and tumours which were already resistant to osimertinib or sotorasib, TT125-802 could still delay cell or tumor growth. Complementary and longitudinal analysis of transcriptional changes using RNA sequencing in vitro and in vivo identified several early adaptive and late acquired resistance signatures that were reversed by TT125-802. A first-in-human study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Thomas Bohnacker, Dorothea Gruber, Sara Laudato, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor. [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 3907.

Abstract 3853: The TWIST1-p27 signaling pathway is a critical determinant of MET TKI cell cycle arrest and resistance in MET altered non-small cell lung cancer

Abstract Advancement in classifying NSCLC into molecularly defined subgroup that respond to specific therapies have shifted the treatment paradigm towards personalized targeted therapy approach. A major obstacle for the treatment of oncogene driven non-small cell lung cancer (NSCLC) patients is both de novo and acquired resistance to targeted therapies. Approximately 4-6% of NSCLC patients harbor MET amplifications/mutations which are important targetable oncogenic drivers in the clinic. In addition, MET amplification has also emerged as a common mechanism of acquired resistance to EGFR TKIs as well as other targeted therapies. Crizotinib, tepotinib and capmatinib are FDA approved MET TKIs for MET exon 14 skipping mutant NSCLC and have shown activity against MET amplified NSCLC as well. Despite high response rates with these newer TKIs, the majority of patients with targetable MET alterations fail to respond and acquired resistance is inevitable. Multiple molecular mechanisms of MET TKIs resistance encompassing both on-target (HGF overexpression) and bypass have been identified. Furthermore, at the time of resistance, an epithelial-to-mesenchymal (EMT) phenotype is often observed. We have found that HGF/MET pathway is a major regulator of the TWIST1, TWIST1 is required for MET altered NSCLC and cooperatives with HGF in vivo during tumorigenesis. Here, we reported that EMT transcription factor TWIST1 mediates a bypass of a MET TKI-mediated p27-dependent growth arrest. To study the mechanism of TKI resistance in MET altered NSCLC, we used multiple MET dysregulated human cell lines (H1437, H596, H1993, and H1648), a novel acquired resistance model of MET TKI resistance (H1993-CR cells), two syngeneic tobacco carcinogen induced mouse lung cell lines (FVBW-17 and FVBCH-17), and three different animal models (mouse xenografts, PDXs, transgenic animals). We found that TWIST1 overexpression led to MET TKI resistance in vitro and in vivo in MET altered cell lines by overcoming cell cycle arrest. This was accompanied by a failure to induce p27 in the presence of TWIST1 overexpression. Similarly, loss of p27 induction and G1 arrest was also observed in our model of acquired MET TKI resistance. Conversely, TWIST1 inhibition restore sensitivity to TKIs in vitro and in vivo and lead to induction of p27. p27 appears to be require for MET TKI induced cell cycle arrest and sensitivity as silencing p27 lead to MET TKI resistance. Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in MET-driven NSCLC. Citation Format: Vinod Kumar, Zachary A. Yochum, Princey Devadassan, Eric Huang, Ethan Miller, Vasavi Ayyala, Purva H. Rumde, Roja Baruwal, James O’Brien, Laura P. Stabile, Timothy F. Burns. The TWIST1-p27 signaling pathway is a critical determinant of MET TKI cell cycle arrest and resistance in MET altered non-small cell lung cancer. [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 3853.

Abstract 99: Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance

Abstract Tumors frequently acquire resistance to therapy resulting in disease relapse and patient mortality. Therapeutic approaches to overcome acquired resistance are hindered by limited treatment options and the presence of multiple parallel resistance mechanisms. Rather than treating resistance after it emerges, it may possible to prevent it by inhibiting the adaptive processes which initiate resistance. However, these processes and the genes which control them are poorly understood. Here we report that drug-tolerant cancer persister cells, which constitute residual disease and seed relapse, undergo drug stress-induced sublethal apoptotic signaling resulting in activation of apoptotic DNase DFFB without cell death. DFFB induces DNA damage and mutagenesis in persister cells, revealing DFFB as a key regulator of stress-induced mutagenesis in persister cells. Furthermore, we found in multiple tumor types that DFFB is required for acquired resistance to targeted therapies. Mechanistically, DFFB induces multiple putative resistance mutations and may also promote epigenetic changes which result in resistance. These results reveal DFFB as a promising potential therapeutic target to prevent acquired resistance. In addition, this stress-sensing mutagenic mechanism may promote mutagenesis in other contexts including normal tissues because sublethal apoptotic signaling may result from a variety of physiological stresses. Citation Format: August Finley Williams, David Gervasio, Claire Turkal, Anna Stuhlfire, Michael Wang, Brandon Mauch, Ariel West, Michelle Paw, Mehrshad Hairani, Cooper Lathrop, Sophie Harris, Jennifer Page, Matthew Hangauer. Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance [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 99.

Abstract 552: Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC

Abstract Despite inducing strong and durable remissions, inhibitors of mutant ALK (ALKi) are not curative for advanced ALK+ lung cancers, as residual tumors eventually develop resistance and relapse. Apart from tumor cells’ intrinsic ability to escape from therapy, there is a growing body of evidence suggesting the contribution of extrinsic factors, produced by cancer-associated fibroblasts (CAFs). Despite the multitude of studies that demonstrated the ability of multiple CAF-produced factors to reduce the sensitivity of tumor cells to ALKi, the contribution of these effects toward responses in vivo remains unresolved. To study the impact of stroma on the ability of tumor cells to survive and develop resistance to ALKi, we derived multiple isolates of fibroblasts from clinical samples. Consistent with previous reports, we found that co-culture with CAFs or CAF-conditioned media protects tumor cells against ALKi. We observed that the degree of protection varies between different CAF isolates. This variability in the extent of protection could be attributed to variability in the levels of secreted hepatocyte growth factor (HGF) a known paracrine mediator of environmental resistance. Moreover, exogenous HGF phenocopied the effect of CAFs while blocking HGF-cMET signaling with neutralizing antibodies or pharmacologically abrogated the protective effect. To test the relevance of these findings in vivo, we took advantage of the inability of murine HGF to activate human cMET by comparing the response of ALK+ xenograft tumors to front-line ALKi alectinib between NSG mice and NSG-derivative strain with humanized HGF. In contrast to the in vitro data, HGF status had only a minimal impact on the remission-relapse dynamics of xenograft tumors. This lack of differences reflected a strong HGF-independent sheltering effect of the stromal niche. Our histological analyses of samples at different points of remission-relapse response revealed that while alectinib potently suppressed tumor cell proliferation, proximity to stroma reduced this cytostatic effect, without impacting cell proliferation in the absence of therapy. To gain insights into the mechanistic underpinning of this HGF-independent effect, we used spatial transcriptomics, comparing stroma-proximal and stroma-distant tumor regions. These analyses, as well as functional validation studies, indicate that stroma-sheltering effects are mediated by multiple mechanisms, acting in an additive fashion. In summary, our studies indicate that therapy resistance of tumor cells reflects the combined action of both intrinsic and microenvironmental factors. Our findings indicate that focusing on a single resistance mechanism at a time is unlikely to induce strong, durable responses. Instead, tackling the issue of therapy resistance necessitates the consideration of multiple resistance mechanisms as well as the moving target nature of tumors under therapy. Citation Format: Bina Desai, Tatiana Miti, Daria Miroshnychenko, Viktoriya Marusyk, Chandler Gatenbee, Menkara Henry, Uwe Rix, Alexander Anderson, Eric Haura, David Basanta, Andriy Marusyk. Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC [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 552.

Abstract 1624: eIF4E allosteric regulators cause rapid commitment to apoptosis in cancer cells while sparing immune cells

Abstract Advanced or metastatic breast cancers represents a large patient population. Targeted therapies following hormonal or chemotherapeutic approaches offer some benefit. Subsequently, molecular profiling has identified over 100 mutations that render inhibitor resistance, creating challenges for physicians, patients, and the healthcare system broadly. While advances in therapeutic options have improved treatment outcomes by prolonging PFS with better QOL, long-term solutions remain a challenge for the majority of patients. The aim of our work is to drive strong mechanistic responses in this large patient population by developing targeted therapies to a key fundamental mechanism at the convergence of many oncogenic signaling pathways through which resistance arises. Our work in established cell lines and primary patient derived organoids demonstrated potent and complete responses in breast cancers that represent patient populations where unmet needs remain high including ER positive, TNBC, PTEN and RB null molecular profiles. The regulation of CAP dependent protein translation is a central element in oncogenesis. One of a family of CAP binding proteins, eIF4E serves to anchor the m7GTP CAP of RNA into several protein complexes and regulates multiple aspects of mRNA processing such as nuclear export and ribosomal translation. Previous communications revealed our eIF4E regulators potently cause cell death in primary breast cancer organoid models differentially to models derived from healthy tissue. We have expanded our efforts following our observations of both rapid apoptosis and differential sensitivity of healthy cells. In vitro, in breast cancer cells we observed stable responses in mechanistic markers of pharmacodynamic response, including phospho-Rb, with short term drug incubation. In vivo, in a ZR-75-1 breast CDX model, intermittent dosing (QW) of our lead compound resulted in strong tumor growth inhibition (>95%). Consistent with growth impacts, we observed stable pharmacodynamic marker (p-Rb) changes in tumor tissue at 24 and 48 hours. To understand the impact on immune cells, we evaluated our regulators against primary cells including PBMCs, CD4+ T cells, and bone marrow mononuclear cells. Our eIF4E regulators did not impair survival of these cells up to a maximum tested dose of 25 micromolar. Primary CD4+ T cells were also functional in the presence of eIF4E regulators up to 5 micromolar, which included assessments of CD3/CD28 stimulated cytokine production and proliferative responses. Our findings suggest that allosteric modulation of eIF4E provides a unique and efficient way to address multiple resistance mechanisms while sparing normal immune cellular function. Thus, our allosteric eIF4E regulators represent a potential beneficial therapeutic approach to address multiple resistant cancer patient populations and fulfill the promise of this elusive target. Citation Format: Lisa-Marie Sturla, Xuemei Zhang, Katherine Bowdish, Susan E. Alters, Christopher VanDeusen. eIF4E allosteric regulators cause rapid commitment to apoptosis in cancer cells while sparing immune cells [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 1624.

CRGD-targeted heparin nanoparticles for effective dual drug treatment of cisplatin-resistant ovarian cancer.

Resistance to the chemotherapeutic agent cisplatin (DDP) is the primary reason for invalid chemotherapy of ovarian cancer. Given the complex mechanisms underlying chemo-resistance, the design of combination therapies based on blocking multiple mechanisms is a rationale to synergistically elevate therapeutic effect for effectively overcoming cancer chemo-resistance. Herein, we demonstrated a multifunctional nanoparticle (DDP-Ola@HR), which could simultaneously co-deliver DDP and Olaparib (Ola, DNA damage repair inhibitor) using targeted ligand cRGD peptide modified with heparin (HR) as nanocarrier, enabling the concurrent tackling of multiple resistance mechanisms to effectively inhibit the growth and metastasis of DDP-resistant ovarian cancer. In combination strategy, heparin could suppress the function of multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) to promote the intracellular accumulation of DDP and Ola by specifically binding with heparanase (HPSE) to down-regulate PI3K/AKT/mTOR signaling pathway, and simultaneously served as a carrier combined with Ola to synergistically enhance the anti-proliferation ability of DDP for resistant ovarian cancer, thus achieving great therapeutic efficacy. Our DDP-Ola@HR could provide a simple and multifunctional combination strategy to trigger an anticipated cascading effect, thus effectively overcoming the chemo-resistance of ovarian cancer.

Laboratory evaluation of broflanilide (TENEBENAL™) against Anopheles gambiae in Moshi, Tanzania – delayed mortality, cross-resistance, and residual efficacy

IntroductionBroflanilide is a novel meta-diamide insecticide, which has a distinct mode of action compared to other active ingredients used for Indoor Residual Spraying (IRS). This study details a laboratory evaluation of broflanilide, of putative discriminating concentrations, potential cross-resistance, and residual efficacy on two substrates.MethodsMosquitoes were exposed to broflanilide in bottle bioassays to determine lethal concentrations (LC50 and LC95). These were used to calculate resistance ratios between the susceptible Kisumu and the pyrethroid-resistant Muleba-Kis strains of Anopheles gambiae s.s. Prototype wettable powder formulations of broflanilide were applied to mud and concrete to determine the optimal observation period for determining delayed mortality, and a Cox proportional hazards model was fitted to determine a potential dose response effect. Subsequently, residual efficacy of application rates ranging between 25 mg/m2 and 300 mg/m2 were monitored monthly.ResultsLC values of tested strains were not significantly different; therefore, the polyfactorial resistance mechanisms possessed by the resistant strain did not confer resistance to broflanilide. A significant effect of concentration and time since spraying was found on mosquito survival, indicating that higher broflanilide concentrations are more effective and that this effect was strongest immediately after spraying. Knockdown at 60min post exposure was negligible, with on average only 1% of all mosquitoes knocked down. On the mud surface, but not on concrete, there was a delayed killing effect, with mortality increasing until 72 hours after exposure. The residual efficacy test indicated that on concrete the 100 mg/m2 and 200 mg/m2 concentrations of both broflanilide 50WP formulations remained efficacious for 9 months post spraying. On mud, there were large variations in mosquito mortality from month to month. Generally, higher concentrations resulted in higher mortality, despite variation over time.DiscussionCross-resistance to broflanilide was not detected in mosquitoes with multiple resistance mechanisms. This opens up the possibility for wide-scale use of broflanilide, especially in areas with established pyrethroid resistance. Like some other insecticide classes, delayed mortality up to 72 hours post exposure was found for broflanilide. Promising residual efficacy was found with broflanilide 50WP formulations on concrete. On mud, efficacy varied and further testing with a refined formulation is recommended.

Treatment Strategies for KRAS-Mutated Non-Small-Cell Lung Cancer.

Activating mutations in KRAS are highly prevalent in solid tumours and are frequently found in 35% of lung, 45% of colorectal, and up to 90% of pancreatic cancers. Mutated KRAS is a prognostic factor for disease-free survival (DFS) and overall survival (OS) in NSCLC and is associated with a more aggressive clinical phenotype, highlighting the need for KRAS-targeted therapy. Once considered undruggable due to its smooth shallow surface, a breakthrough showed that the activated G12C-mutated KRAS isozyme can be directly inhibited via a newly identified switch II pocket. This discovery led to the development of a new class of selective small-molecule inhibitors against the KRAS G12C isoform. Sotorasib and adagrasib are approved in locally advanced or metastatic NSCLC patients who have received at least one prior systemic therapy. Currently, there are at least twelve KRAS G12C inhibitors being tested in clinical trials, either as a single agent or in combination. In this study, KRAS mutation prevalence, subtypes, rates of occurrence in treatment-resistant invasive mucinous adenocarcinomas (IMAs), and novel drug delivery options are reviewed. Additionally, the current status of KRAS inhibitors, multiple resistance mechanisms that limit efficacy, and their use in combination treatment strategies and novel multitargeted approaches in NSCLC are discussed.

Abstract P1-13-14: Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, and despite recent treatment advances, clinical outcomes remain poor, particularly in the metastatic setting. TNBC is a biologically heterogeneous entity, and up to 50% of TNBC express the androgen receptor (AR). Gene expression subtyping has identified a subset of AR-TNBC with a luminal AR-driven (LAR) phenotype. Consequently, there has been great interest in translating androgen receptor signaling inhibitors (ARSIs) approved for use in prostate cancer such as enzalutamide and bicalutamide into the management of LAR-TNBC. However, early phase clinical trials of anti-androgens in metastatic AR-TNBC have had modest results. While multiple mechanisms of anti-androgen resistance have been identified in prostate cancer, including AR amplification and over-expression, emergence of AR splice variants (AR-Vs), and transition to AR independent growth, molecular determinants of anti-androgen resistance in TNBC remain poorly understood, and pre-clinical models to study acquired ARSI resistance in LAR-TNBC have been limited. Methods: A novel pre-clincial model of acquired ARSI resistance was derived from the LAR subtype MDA-MB-453 cell line through serial passaging with increasing concentrations of enzalutamide. Celltiter Blue viability assays were used to determine enzalutamide IC50 of parental and enzalutamide-resistant (EnzR) cell lines. RNA was extracted from the parental and resistant cells, reverse transcribed and expression of AR, splice-variants and canonical AR target genes were evaluated using RT-qPCR. Immunofluorescence was used to investigate the amount and nuclear localization of the AR protein within the parental and enzalutamide resistant cells. Results: EnzR MDA-MB-453 cells were derived through culture in increasing concentrations of enzalutamide for >6 months, and found to have a significant increase in enzalutamide IC50 compared to the parental cell line. EnzR MDA-MB-453 cells had a 4.5 fold increase in full-length AR expression at the transcriptional level compared to the parental cell line. AR protein expression and nuclear localization were also increased in EnzR cells compared to the parental cell line. Canonical AR targets including NKX3.1 were downregulated in response to enzalutamide in parental but not EnzR cells. While pathogenic AR splice variants (AR-Vs) implicated in ARSI resistance in prostate cancer were detected at low levels in MDA-MB-453 cells, no increase in AR-V expression was observed in the EnzR cell line. Conclusions: We have developed a novel pre-clinical model of anti-androgen/ARSI resistance in LAR-TNBC, and identified AR overexpression and persistent AR signaling associated with acquired enzalutamide resistance in LAR-TNBC. While pathogenic AR splice variants have been implicated in ARSI resistance in metastatic prostate cancer and are detected in the MDA-MB-453 cell line model, enzalutamide resistance was not associated with increased AR splice variant expression in this model. Future work will investigate mechanisms leading to AR overexpression as well as combination therapeutic strategies to overcome acquired ARSI resistance. Citation Format: Hannah Krause, Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Kari B. Wisinski, Ruth O’Regan, Joshua Lang. Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer. [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 P1-13-14.