Activation Of Compensatory Pathways Research Articles

Anti-EGFR antibody-drug conjugate carrying an inhibitor targeting CDK restricts triple-negative breast cancer growth.

Anti-EGFR antibodies show limited response in breast cancer, partly due to activation of compensatory pathways. Furthermore, despite clinical success of CDK4/6 inhibitors in hormone receptor-positive tumors, aggressive triple-negative breast cancers (TNBCs) are largely resistant due to CDK2/cyclin E expression, while free CDK2 inhibitors display normal tissue toxicity, limiting their therapeutic application. A cetuximab-based antibody drug conjugate (ADC) carrying a CDK inhibitor selected based on oncogene dysregulation, alongside patient subgroup stratification, may provide EGFR-targeted delivery. Expression of G1/S-phase cell cycle regulators were evaluated alongside EGFR in breast cancer. We conjugated cetuximab with CDK inhibitor SNS-032, for specific delivery to EGFR-expressing cells. We assessed ADC internalization, and its anti-tumor functions in vitro and in orthotopically-grown basal-like/TNBC xenografts. Transcriptomic (6173 primary, 27 baseline and matched post-chemotherapy residual tumors), scRNA-seq (150290 cells, 27 treatment-naïve tumors) and spatial transcriptomic (43 tumor sections, 22 TNBCs) analyses confirmed expression of CDK2 and its cyclin partners in basal-like/TNBCs, associated with EGFR. Spatiotemporal live-cell imaging and super-resolution confocal microscopy demonstrated ADC colocalization with late lysosomal clusters. The ADC inhibited cell cycle progression, induced cytotoxicity against high EGFR-expressing tumor cells and bystander killing of neighboring EGFR-low tumor cells, but minimal effects on immune cells. Despite carrying a small fraction of the drug, the ADC restricted EGFR-expressing spheroid and cell line/patient-derived xenograft tumor growth. Exploiting EGFR overexpression, and dysregulated cell cycle in aggressive and treatment-refractory tumors, a cetuximab-CDK inhibitor ADC may provide selective and efficacious delivery of cell cycle-targeted agents to basal-like/TNBCs, including chemotherapy-resistant residual disease.

Abstract 228: Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients

Abstract Glioblastoma (GBM) is the most common and incurable adult primary brain tumour. Treatment resistance and relapse is the primary cause of death in GBM. Tumour heterogeneity is one of the significant determinants of treatment failure. Patient-derived cancer organoids have been shown to maintain genetic and phenotypic tumour features and recapitulate intratumoral heterogeneity. Here, we report the development and molecular characterisation of patient-derived GBM organoid (PGO) models to study treatment response to existing and new treatment combinations. GBM biopsies were dissociated, suspended in Cultrex, and cultured in a brain stem cell medium. PGOs were characterised by whole-exome (WES) single-cell karyotype (scKaryoSeq) and RNA sequencing. The IC-50 and viability of PGO upon temozolomide (TMZ) and small molecule inhibitors were determined. Gene expression profiling was performed before and after TMZ treatment. Single-cell spatial heterogeneity was determined using Multiplex Immunostaining of PGO and matched patient samples. PGOs were established with a success rate of 80% (n=31). WES analysis between PGOs from the same patient at multiple time points (1-3 months) showed high maintenance of Single Nucleotide Variants (92.3%-97.7%). In PGOs key somatic oncogene variants and copy number variations were retained compared to the primary tumour (58%-90%). scKaryoseq (N=7) of different clinical GBM subtypes showed a high degree of genetic heterogeneity, with some PGO having up to 10 subclones. Single-cell phenotypic landscaping of patient biopsies showed mixed cellular states maintained after long-term culture in the matched PGO. PGOs showed a differential response to TMZ, corresponding with MGMT methylation and patient mutation status. RNAseq of PGO revealed TMZ-responsive genes, including upregulation of the prognostic JUN kinase pathway. Combination treatment of a JUN kinase inhibitor and TMZ showed a synergistic effect. In addition, PGOs were screened for their sensitivity to clinically approved brain-penetrable small molecule inhibitors targeting key GBM driver pathways. Combining small molecular inhibitors of MEK/PI-3/CDK4/EGFR kinase strongly reduced cell viability and activation of cell death pathways. phosphoKinome screens detected compensatory pathway activation upon kinase inhibition (MEK/CDK4 inhibitor) in several PGO. Data will show novel kinase interaction pathways that increase drug sensitivity in PGO. These data show that PGOs can be used as a stable phenotypic and genetic representative of the patient's tumour and have the potential to function as patient avatars for adaptive treatment selection and actionable drug target discovery. Citation Format: Marc A. Vooijs, Linde Hoosemans, Jolanda Piepers, Maikel Verduin, Maxime Vanmechelen, Mike Van Heumen, Frederik De Smet, Ann Hoeben. Patient-derived gbm organoids reflect tumour heterogeneity and treatment sensitivity in patients [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 228.

SHP2 inhibitors maintain TGFβ signalling through SMURF2 inhibition

Despite the promising antitumor activity of SHP2 inhibitors in RAS-dependent tumours, overall responses have been limited by their narrow therapeutic window. Like with all MAPK pathway inhibitors, this is likely the result of compensatory pathway activation mechanisms. However, the underlying mechanisms of resistance to SHP2 inhibition remain unknown. The E3 ligase SMURF2 limits TGFβ activity by ubiquitinating and targeting the TGFβ receptor for proteosome degradation. Using a functional RNAi screen targeting all known phosphatases, we identify that the tyrosine phosphatase SHP2 is a critical regulator of TGFβ activity. Specifically, SHP2 dephosphorylates two key residues on SMURF2, resulting in activation of the enzyme. Conversely, SHP2 depletion maintains SMURF2 in an inactive state, resulting in the maintenance of TGFβ activity. Furthermore, we demonstrate that depleting SHP2 has significant implications on TGFβ-mediated migration, senescence, and cell survival. These effects can be overcome through the use of TGFβ-targeted therapies. Consequently, our findings provide a rationale for combining SHP2 and TGFβ inhibitors to enhance tumour responses leading to improved patient outcomes.

The activity of a PI3K δ-sparing inhibitor, MEN1611, in non-small cell lung cancer cells with constitutive activation of the PI3K/AKT/mTOR pathway.

Lung cancer remains the leading cause of cancer-related death worldwide. Targeted therapies with tyrosine kinase inhibitors (TKIs) result in improvement in survival for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR). Unfortunately, most patients who initially respond to EGFR-TKI ultimately develop resistance to therapy, resulting in cancer progression and relapse. Combination therapy is today a common strategy for the treatment of tumors to increase the success rate, improve the outcome and survival of patients, and avoid the selection of resistant cancer cells through the activation of compensatory pathways. In NSCLC, the phosphoinositide-3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway has been heavily implicated in both tumorigenesis and the progression of disease. In this study, we investigated the efficacy of a PI3K δ-sparing inhibitor, MEN1611, in models of NSCLC sensitive and resistant to EGFR inhibitors (erlotinib and gefitinib) with a wild-type PIK3CA gene. We performed functional, biochemical, and immunohistochemistry studies. We demonstrated good efficacy of MEN1611 in NSCLC devoid of PIK3CA gene mutations but with constitutive activation of the PI3K/AKT pathway and its synergistic effect with gefitinib both in vitro and in vivo. Overall, this preclinical study indicates that the inhibitor could be a candidate for the treatment of NSCLC with an erlotinib/gefitinib-resistant phenotype and constitutive activation of the PI3K/AKT pathway, a phenotype mimicked by our model system.

A phase I trial of sequential dosing of sonidegib and pembrolizumab in advanced solid tumors (aST) and non–small-cell lung cancer (NSCLC).

9093 Background: Pembrolizumab interrupts PD-1/-L1 interaction and is efficacious in many cancers. However, resistance invariably emerges in the majority of patients. Potential mechanisms include activation of compensatory pathways. The inhibition of sonic hedgehog signaling (sHH) was previously shown to induce intra-tumoral (IT) infiltration by CD4+, CD8+ and HLA-DR class II cells. Our preclinical data showed sHH inhibitor enhanced IT penetration by monoclonal antibodies and induced PD-L1 expression. We conducted a phase I study using a novel sequential pulse dosing of sonedegib followed by pembrolizumab in aST with an expansion cohort to determine the maximum tolerated dose (MTD), tolerability and preliminary efficacy. Methods: The dose escalation utilized a 3+3 design to determine the MTD and proceeded to expansion cohort upon determination of the MTD. Patients enrolled to each dose level were observed for dose limiting toxicities (DLTs) during the 1st cycle. The expansion cohort consisted of NSCLC and head/neck squamous cell carcinoma (HNSCC) previously treated by anti-PD1/-L1. The recommended phase 2 dose (RP2D) was determined following the review of the toxicity profile of the combination from all cycles. The study treatment consisted of sonidegib oral daily from day 1 to 8 and pembrolizumab I.V. 200 mg on day 8 on an every 3-week cycle. The sonidegib dose levels explored were 400, 600 and 800 mg. Archival tumor and blood specimens were collected for correlative studies. Adverse events (AEs) were categorized and graded per CTCAE v5.0, and tumor response by RECIST v1.1. Results: The study enrolled a total of 33 patients and 29 received treatment (13 in escalation and 16 in expansion), including patients with NSCLC, HNSCC, skin cancer, gastric cancer and melanoma. The enrollment to the NSCLC cohort is complete and HNSCC continues. No DLT was encountered during dose escalation and the MTD was not reached. The highest planned dose level was selected to proceed to expansion (sonidegib 800mg days 1-8 in combination with pembrolizumab I.V. 200 mg on day 8 on an every 3-week cycle). The expansion cohort proceeded to enroll 15 patients evaluable for AEs (10 NSCLC and 5 HSNCC). Grade 3 and above treatment-related AEs across all patients were hyperglycemia, fatigue, dyspepsia, abdominal pain. The mature efficacy data will be reported at the meeting. Conclusions: The study successfully determined the RP2D of sonidegib when dosed sequentially with pembrolizumab in advanced solid tumors. The AE profile of the combination is similar to that for respective agents given individually. The efficacy data among anti-PD1/-PDL1-treated NSCLC will be reported at the meeting. Clinical trial information: NCT04007744 .

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 3856: Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy

Abstract Lung cancer is the leading cause of cancer related deaths worldwide with the majority (~80%) of patients diagnosed with non-small lung cancer (NSCLC). Targeted therapies directed against tumor initiating growth factor receptors are effective treatment options for driver mutation positive NSCLC. Osimertinib is a third-generation EGFR-Tyrosine Kinase inhibitor (TKI) that has activity against EGFR exon 19, exon 21, and T790M mutations. Unfortunately, Osimertinib treated patients acquire resistance and median progression free survival is 18 months. Moreover, Osimertinib does not cure stage IV EGFR mutant NSCLC. The acquisition of secondary mutations leading to activation of bypass pathways, MET amplification, EMT, compensatory pathway activation and histological transformation to small cell phenotype are major causes of acquiring resistance to EGFR TKIs. Proliferating cancer cells regularly experience a low level of replication stress. Additionally, receptor tyrosine kinases such as EGFR are known to interact with DNA repair proteins and impact DNA damage repair following chemotherapy, radiation therapy, and EGFR TKI treatment. However, the role of DNA repair pathways in EGFR-TKI resistance is unknown and the interaction between EGFR pathway, DNA damage, and repair pathways has not been fully elucidated. At low-level replicative stress promotes genomic instability but at a high level through mitotic catastrophe it causes cell death. Replication protein A is a critical sensor of the DNA damage response detecting replication stress. The small molecule inhibitor of RPA, NERx 329, sequesters active RPA and induces replication catastrophe and cell death. Here we demonstrate that NERx329 in combination with Osimertinib enhanced Osimertinib mediated cell death. CCk-8 assays performed after 48h of NERx329 and Osimertinib combination treatment result in a robust decrease in IC50 compared to single agent therapy. Dissection of signaling pathways leading to this death-promoting effect of NERx329 showed a combination of bypass pathway marker (AXL) and compensatory pathway marker (STAT) inhibition in AXL-high expressing EGFR mutant lung cancer cells. Whereas inhibition of STAT pathway was responsible for the death-promoting effect of NERx329 in AXL-low expressing EGFR mutant lung cancer cells. AXL and STAT inhibition are associated with induced DNA damage and impaired DNA repair. From these data, we infer that DNA damage repair pathways could be involved in TKI resistance and NERx329 could be a promising drug candidate for combination targeted therapy. Citation Format: Reshma Bhowmick, John J. Turchi, Shadia I. Jalal. Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy. [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 3856.

174 (PB054) - A novel BAD phosphorylation inhibitor combined with EGFR tyrosine kinase inhibitors in EGFR-mutant lung adenocarcinoma treatment

Background: The approval and use of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in EGFR-mutant NSCLC has been a breakthrough in clinic. The activation of compensatory pathways of PI3 K/ AKT and/or MEK/MAPK modulates cell survival and drug resistance of lung adenocarcinoma (LUAC). As a convergent downstream signaling node and the determinant, BAD is a potential target to inhibit cell survival and to overcome EGFR TKI resistance in LUAC treatment.

Intermittent MEK inhibition for the treatment of metastatic uveal melanoma.

IntroductionUveal melanoma (UM) is associated with poor outcomes in the metastatic setting and harbors activating mutations resulting in upregulation of MAPK signaling in almost all cases. The efficacy of selumetinib, an oral allosteric inhibitor of MEK1/2, was limited when administered at a continual dosing schedule of 75 mg BID. Preclinical studies demonstrate that intermittent MEK inhibition reduces compensatory pathway activation and promotes T cell activation. We hypothesized that intermittent dosing of selumetinib would reduce toxicity, allow for the administration of increased doses, and achieve more complete pathway inhibition, thus resulting in improved antitumor activity.MethodsWe conducted a phase Ib trial of selumetinib using an intermittent dosing schedule in patients with metastatic UM. The primary objective was to estimate the maximum tolerated dose (MTD) and assess safety and tolerability. Secondary objectives included assessment of the overall response rate (RR), progression-free survival (PFS) and overall survival (OS). Tumor biopsies were collected at baseline, on day 3 (on treatment), and between days 11-14 (off treatment) from 9 patients for pharmacodynamic (PD) assessments.Results29 patients were enrolled and received at least one dose of selumetinib across 4 dose levels (DL; DL1: 100 mg BID; DL2: 125 mg BID; DL3: 150 mg BID; DL4: 175 mg BID). All patients experienced a treatment-related adverse event (TRAE), with 5/29 (17%) developing a grade 3 or higher TRAE. Five dose limiting toxicities (DLT) were observed: 2/20 in DL2, 2/5 in DL3, 1/1 in DL4. The estimated MTD was 150 mg BID (DL3), with an estimated probability of toxicity of 29% (90% probability interval 16%-44%). No responses were observed; 11/29 patients achieved a best response of stable disease (SD). The median PFS and OS were 1.8 months (95% CI 1.7, 4.5) and 7.1 months (95% CI 5.3, 11.5). PD analysis demonstrated at least partial pathway inhibition in all samples at day 3, with reactivation between days 11-14 in 7 of those cases.ConclusionsWe identified 150 mg BID as the MTD of intermittent selumetinib, representing a 100% increase over the continuous dose MTD (75 mg BID). However, no significant clinical efficacy was observed using this dosing schedule.

Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood-Brain Barrier and Target EGFR/PI3Kp110β Signaling.

Simple SummaryTreatment of glioblastoma is hampered by the activation of compensatory survival mechanisms by malignant cells that lead to drug resistance. Moreover, the blood–brain barrier (BBB) precludes the brain entrance of most drugs. We hypothesized that computer-assisted drug discovery tools would reveal novel multi-targeting drug candidates with BBB-permeant and favorable ADMET properties. We aimed to discover molecules with predicted ability to inhibit the EGFR/PI3Kp110β pathway and to validate their efficacy and safety in biological assays. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Moreover, we tested their anti-tumor efficacy and BBB safety and permeation in cell models. We found two EGFR, two PI3Kp110β, and, mostly, two dual inhibitors with anti-tumor effects. Among them, one EGFR and two PI3Kp110β inhibitors were able to cross the BBB endothelium without compromising it. These studies revealed novel drug candidates for glioblastoma treatment.The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110β signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood–brain barrier (BBB) limits drugs’ brain penetration. We aimed to discover EGFR/PI3Kp110β pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110β, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110β, and two dual inhibitors) decreased cell viability by 40–99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets’ downregulation and increased apoptosis (15–85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110β inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.

Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer.

In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called 'TRIM-ing'. To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines. Cancer cells were treated with HER2-targeted medicines (Trastuzumab and Neratinib) or STAT3 targeted inhibitors (Stattic and Niclosamide) with or without respective target TRIM-ing. Real-time PCR, immunoblotting, immunofluorescence, cytotoxicity, short- and long-term cell survival assessments were done following standard methodologies. 3-D structure modelling was used to verify the binding of mAb onto the STAT3 target. TRIM-ing of HER2 or HER3 receptors or their activated phospho-forms in BC cells showed rapid degradation of respective protein forms, shattering down the downstream signaling (p-ERK, p-AKT) that lasts for up to 7-8days. This significantly inhibited BC survival (p < 0.001), showing a synergistic therapeutic effect with HER2 medicine trastuzumab or neratinib. Additionally, specific TRIM-ing ability of canonical pY705 or non-canonical pS727 PTMs of STAT3 protein was demonstrated in MCF7 cells, causing significant cytotoxicity (p < 0.05). TRIM-ing of STAT3 PTM, when combined with the same PTM-specific inhibitors, a synergistic treatment effect was observed. The work demonstrated that TRIM-ing could directly reduce various oncogenic targets or their specific activated form inside the cancer cells without compensatory pathway activation, a conundrum limiting the therapeutic benefit of current personalized medicines.

Abstract 5211: Therapeutic resistance to anti-AR signal pathway therapies is related to compensatory pathway activation and immune suppression in mouse Pten/Trp53-deficient CRPC

Abstract Prostate cancers depend on androgens and the androgen receptor (AR) to drive androgen signaling pathways. Androgen-deprivation therapy (ADT) and second-generation antiandrogens have become the new standard of care for men with prostate cancer. However, resistance to these therapies remains a problem. TP53 mutations and PTEN loss have been associated with patients who fail to respond to anti-androgen therapies and complex interactions between AR signaling and the immune system means that these therapies have the potential to affect antitumor immune responses. Here, we characterize treatment responses of ADT alone and in combination with antiandrogen therapy using apalutamide (Apa) in a mouse model of Pten/Trp53-deficeint castrate-resistant prostate cancer (CRPC). Twenty-eight-week-old conditional Pten/Trp53-double knockout mice were randomized as untreated control or treated with ADT via orchidectomy for four weeks followed by vehicle or Apa therapy for an additional four weeks. While both treatments reduced tumor burden, there was no difference between treatments. Treatment responses in were further evaluated and were judged as favorable or non-favorable, relative to median tumor burden. Analysis of individual treatment responses indicated that 31.3% (5/16) of mice treated with ADT+Apa had tumor burden reductions &amp;gt;20% (relative to the median) compared to 6.3% (1/16) in ADT, and in both instances, 37.5% (6/16) of mice treated with ADT or ADT+Apa had tumor burden increases &amp;gt;20%. IHC showed lower nuclear/cytoplasm expression of AR in ADT+Apa treated mice and the reduction was more pronounced in favorable responders. ADT+Apa treated mice had lower gene expression levels of Ar than ADT alone and AR target genes (Fkbp5, Tmprss2, Timp4 and Nkx3.1) were decreased in ADT+Apa favorable responders whereas these target genes as well as AR-regulating genes (Myc and Igf1r, Igf1) were higher in ADT+Apa non-favorable responders. Cancer cell proliferation did not differ between treatments, but cancer glands in ADT+Apa treated mice had significantly greater IHC expression levels of cleaved caspase 3, a marker for apoptosis, and were greatest in ADT+Apa favorable responders. Levels of p-S6 ribosomal protein, a downstream target of Akt, were higher in the cancer glands of ADT+Apa but were reduced in stromal and infiltrating immune cells. Immune profiling by gene expression and flow cytometry showed enhanced macrophage involvement and reduced MDSC/granulocyte activity in ADT+Apa treated mice. There were no differences in CD8 T cell and NK cell abundance between the two cohorts, but greater cytotoxic activity was noted in all favorable responders. This study provides preclinical data that links therapeutic resistance to anti-AR signal pathways therapies in Pten/Trp53-deficeint CRPC to compensatory pathway activation and immune suppression. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazutoshi Fujita, Eri Banno, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Therapeutic resistance to anti-AR signal pathway therapies is related to compensatory pathway activation and immune suppression in mouse Pten/Trp53-deficient CRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5211.

Targeting Cell Cycle Progression in HER2+ Breast Cancer: An Emerging Treatment Opportunity.

Layman summaryHER2 is an oncogenic driver in a subset of breast cancer. Despite the fact that there are the options of several anti-HER2 targeted therapies, most patients with metastatic HER2+ breast cancer die from the disease. Therapies to overcome treatment resistance in the metastatic settings (including brain metastasis) are actively being pursued. Recently, cell cycle inhibitors (CDK 4/6 inhibitors) have been approved to manage hormone receptor-positive breast cancer, and have encountered tremendous success. The cell cycle signaling proteins, Cyclin D-CDK4/6, are downstream of HER2 and play a key role in cellular proliferation. Moreover, cell cycle inhibitors have the capacity to cross the blood–brain barrier. Here, we review the published literature with regard to the rationale for CDK4/6-directed therapies in HER2+ breast cancer.The development of HER2-targeted therapies has dramatically improved patient survival and patient management and increased the quality of life in the HER2+ breast cancer patient population. Due to the activation of compensatory pathways, patients eventually suffer from resistance to HER2-directed therapies and develop a more aggressive disease phenotype. One of these mechanisms is the crosstalk between ER and HER2 signaling, especially the CDK4/6-Cyclin D-Rb signaling axis that is commonly active and has received attention for its potential role in regulating tumor progression. CDK 4/6 inhibitors interfere with the binding of cell-cycle-dependent kinases (CDKs) with their cognate partner cyclins, and forestall the progression of the cell cycle by preventing Rb phosphorylation and E2F release that consequentially leads to cancer cell senescence. CDK 4/6 inhibitors, namely, palbociclib, ribociclib, and abemaciclib, in combination with anti-estrogen therapies, have shown impressive outcomes in hormonal receptor-positive (HR+) disease and have received approval for this disease context. As an extension of this concept, preclinical/clinical studies incorporating CDK 4/6 inhibitors with HER2-targeted drugs have been evaluated and have shown potency in limiting tumor progression, restoring therapeutic sensitivity, and may improving the management of the disease. Currently, several clinical trials are examining the synergistic effects of CDK 4/6 inhibitors with optimized HER2-directed therapies for the (ER+/-) HER2+ population in the metastatic setting. In this review, we aim to interrogate the burden of HER2+ disease in light of recent treatment progress in the field and examine the clinical benefit of CDK 4/6 inhibitors as a replacement for traditional chemotherapy to improve outcomes in HER2+ breast cancer.

Role of SOX18 in Promoting Tumorigenesis in Pediatric Cancer Cell Lines

Sarcomas constitute a high percentage (∼13%) of cancer‐related deaths among pediatric patients between 0‐19 years of age, with Rhabdomyosarcoma (RMS) being the most common pediatric soft tissue sarcoma and Ewing Sarcoma being the second most common malignant bone tumor in children. Yet, survival for those who develop such metastatic sarcomas remains below 20‐30%. Interestingly, SOX family proteins are known to be up regulated in various cancer types and play a role in cancer progression (tumorigenesis, metastasis, etc.). More specifically, targeted knockdown of SOX18 has been shown to suppress various tumorigenic properties in cancer cell lines including osteosarcoma cells, hepatocellular carcinoma cells and breast cancer cells. Additionally, prior studies showed inhibition of IGFR leads to compensatory pathway activation via other RTK receptors resulting in continued cell survival. Upregulation of SOX18 was observed in such cases. Thus, it is important to clarify the role of SOX18 in RMS and ES. Ewing Sarcoma (ES8) and Rhabdomyosarcoma (RH41) cell lines were infected with shSOX18 to produce knockdown and selected via puromycin resistance. Western blotting and RT‐qPCR was used to analyze baseline and resultant protein and mRNA levels, respectively after knockdown. Results showed that the shRNA was effective in SOX18 knockdown and lead to reduced cell proliferation. Cell migration, cell proliferation and colony formation assays were used to characterize effects of knockdown. Further evaluation of knockdown effects on angiogenesis was analyzed using chick chorioallantoic membrane (CAM) assays. For ES8 and RH41 cell lines, SOX18 knockdown led to reduced cell migration, cell proliferation and colony formation. We anticipate characterization of the role of SOX18 in RMS and ES may inform future development of novel targeted treatments and increased understanding of role of SOX18 in activation of RTK compensatory pathways during IGFR inhibition in pediatric sarcomas.

Jolkinolide B sensitizes bladder cancer to mTOR inhibitors via dual inhibition of Akt signaling and autophagy.

The monotherapy of mTOR inhibitors (mTORi) in cancer clinical practice has achieved limited success due to the concomitant activation of compensatory pathways, such as Akt signaling and cytoprotective autophagy. Thus, the combination of mTORi and the inhibitors of these pro-survival pathways has been considered a promising therapeutic strategy. Herein, we report the synergistic effects of a natural anti-cancer agent Jolkinolide B (JB) and mTORi (temsirolimus, rapamycin, and everolimus) for the effective treatment of bladder cancer. A mechanistic study revealed that JB induced a dual inhibition of Akt feedback activation and cytoprotective autophagy, potentiating the anti-proliferative efficacy of mTORi in both PTEN-deficient and cisplatin-resistant bladder cancer cells. Meanwhile, mTORi augmented the pro-apoptotic and pro-paraptotic effects of JB by reinforcing JB-activated endoplasmic reticulum stress and MAPK pathways. These synergistic mechanisms were related to cellular reactive oxygen species accumulation. Our study suggests that dual inhibition of Akt feedback activation and cytoprotective autophagy is an effective strategy in mTORi-based therapy, and JB+mTORi combination associated with multiple anti-cancer mechanisms and good tolerance in mouse models may serve as a promising treatment for bladder cancer.

Inhibition of cotranslational translocation by apratoxin S4: Effects on oncogenic receptor tyrosine kinases and the fate of transmembrane proteins produced in the cytoplasm

Receptor tyrosine kinases (RTKs) have become major targets for anticancer therapy. However, resistance and signaling pathway redundancy has been problematic. The marine-derived apratoxins act complementary to direct kinase inhibitors by downregulating the levels of multiple of these receptors and additionally prevent the secretion of growth factors that act on these receptors by targeting Sec61α, therefore interfering with cotranslational translocation. We have profiled the synthetic, natural product-inspired apratoxin S4 against panels of cancer cells characterized by differential sensitivity to RTK inhibitors due to receptor mutations, oncogenic KRAS mutations, or activation of compensatory pathways. Apratoxin S4 was active at low-nanomolar to sub-nanomolar concentrations against panels of lung, head and neck, bladder, and pancreatic cancer cells, concomitant with the downregulation of levels of several RTKs, including EGFR, MET and others. However, the requisite concentration to inhibit certain receptors varied, suggesting some differential substrate selectivity in cellular settings. This selectivity was most pronounced in breast cancer cells, where apratoxin S4 selectively targeted HER3 over HER2 and showed greater activity against ER+ and triple negative breast cancer cells than HER2+ cancer cells. Depending on the breast cancer subtype, apratoxin S4 differentially downregulated transmembrane protein CDCP1, which is linked to metastasis and invasion in breast cancer and modulates EGFR activity. We followed the fate of CDCP1 through proteomics and found that nonglycosylated CDCP1 associates with chaperone HSP70 and HUWE1 that functions as an E3 ubiquitin ligase and presumably targets CDCP1, as well as potentially other substrates inhibited by apratoxins, for proteasomal degradation. By preventing cotranslational translocation of VEGF and other proangiogenic factors as well as VEGFR2 and other receptors, apratoxins also possess antiangiogenic activity, which was validated in endothelial cells where downregulation of VEGFR2 was observed, extending the therapeutic scope to angiogenic diseases.

HDAC6-HSP90 pathway-mediated protein folding and degradation regulates tumor cell proliferation and metastasis

The folding and degradation of proteins in tumor cells are the key mechanisms maintaining their survival, and functions of the proteins involved are often related to the prevailing tumor resistance. With further scientific research, an increasing number of auxiliary proteins involved in protein folding and degradation have been reported in the literature. The chaperone heat shock protein 90 (HSP90) not only promotes protein folding and plays a role in maintaining protein conformation stability but also assists the proteasomal degradation of client proteins. Current research suggests that the function of HSP90 is regulated by post-translational modifications, such as acetylation. Histone deacetylase 6 (HDAC6) can inhibit the over-acetylation of HSP90 and maintain the chaperone function of HSP90 to ensure that the client protein of the chaperone HSP90 can be stably folded and maturely released, which creates conditions for tumor survival, proliferation, and metastasis. Furthermore, it can act as the client protein and autophagy receptor of HSP90. It recruits ubiquitinated proteins and deacetylates and modifies autophagy-related proteins, thus playing important roles in protein degradation and autophagy. Recently, studies revealed HDAC6, a client protein of HSP90, which plays an important role in protein degradation, cell autophagy, and maintenance of the activity of HSP90 by preventing its excessive acetylation. By inhibiting HDAC6 to promote HSP90 over-acetylation, it can effectively inhibit the binding between HSP90 and ATP, thereby affecting the folding and degradation of HSP90 on tumor proliferation and metastasis-related proteins. Simultaneously, HDAC inhibitors can help HSP90 bind more closely to its inhibitors. Therefore, HDAC6 and HSP90 inhibitors have also been widely used in related research on tumor treatment, and studies have shown that the combined application of HDAC6 and HSP90 inhibitors has a significant effect on inhibiting tumor proliferation and metastasis. Whether it is to inhibit HDAC6 alone or HSP90 alone, there are deficiencies in cancer treatment. The use of inhibitors alone may lead to the activation of other pathways related to tumor cell proliferation and survival. HDAC6-HSP90 dual-target inhibition can reduce the activation of compensatory pathways, such as autophagy, and reduce the possibility of off-target effects of HSP90 inhibitors. In this review, we focus on the crosstalk between HDAC6 and HSP90 to further explain how tumor cells initiate proliferation and metastasis signals by regulating protein folding and degradation mechanisms and discuss the research applications of HDAC6 and HSP90 inhibitors. The advantages and disadvantages are assessed to obtain novel strategies for cancer therapy by targeting the HSP90-HDAC6 pathway.

Abstract 6406: FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma

Abstract Uveal melanoma (UM) is characterized by gain-of-function mutations in GNAQ or GNA11, encoding Gα proteins from the Gq/11 family. UM is the most common eye malignancy in adults. Approximately 50% of UM patients develop liver metastasis (mUM) within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. mUM is refractory to cytotoxic, targeted, and immunotherapies, with most mUM patients dying within a year. Recent information suggests that GNAQ-oncogenic signaling involves a non-canonical pathway distinct from the activation of PLCβ and PKC-MEK-ERK, which may explain the failure of MEK inhibitors (MEKi) in increasing mUM patient survival. Instead, we found that GNAQ promotes the activation of YAP1, a key oncogenic driver, by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF (Cancer Cell, 2014). In turn, YAP1 is essential for uveal melanoma cell growth, however no effective and safe YAP1 inhibitors are currently available. Using a novel bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), is a synthetic lethal gene with GNAQ activation, and uncovered that GNAQ controls YAP1 through FAK (Cancer Cell, 2019). This study identified FAK as a druggable signaling hub downstream from GNAQ in UM. However, activation of compensatory pathways often results in resistance to targeted agents. Here, we combined the use of CRISPR-Cas9 sgRNA screens with a recently described Cancer Signaling Toolkit approach to identify synthetic lethal interactions enhancing the response to FAKi and signaling networks mediating drug resistance, respectively. Remarkably, both approaches converged to reveal that co-targeting FAK and the MEK-ERK pathway would be a promising combination for treatment of UM. Indeed, MEK-ERK pathway inhibition by multiple approved MEKis (e.g., trametinib), combined with FAK inhibition (VS-4718 or defactinib), showed remarkable synergistic growth inhibitory effects in UM cells. Additionally, the novel RAF/MEK inhibitor RO5126766 also showed synergistic anti-proliferative effects with defactinib. Accordingly, FAKi combination with MEKi exerted cytotoxic effects (apoptotic death) leading to rapid tumor shrinkage in UM xenografts, whereas single drugs were primarily cytostatic. Furthermore, the FAKi/MEKi combination was successful at reducing tumor burden in recently developed liver metastasis UM models. By coupling the unique genetic landscape of UM with the power of unbiased computational pipelines and systems biology genetic screens, our studies revealed that FAK and MEK-ERK co-targeting may provide a new network-based precision therapeutic strategy for mUM treatment. Indeed, the combination of defactinib and RO5126766 is currently being evaluated in patients with various solid tumors (NCT03875820), and could be explored in mUM based on these preclinical findings. Citation Format: Justine S. Paradis, Monica Acosta, Nadia Arang, Robert Saddawi-Konefka, Ayush Kishore, Takahito Sugase, Xiaodong Feng, Kris C. Wood, Silvia Coma, Mizue Terai, Takami Sato, Jonathan A. Pachter, J. Silvio Gutkind. FAK and MEK co-targeting: A new multimodal precision therapy for GNAQ-driven uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6406.

Abstract 1737: Nanotherapeutic targeting of drug resistant multiple myeloma cells through VLA4-contact facilitated drug-delivery of camptothecin prodrug

Abstract Multiple Myeloma (MM) is a recurring-relapsing plasma cell malignancy, which invariably develops drug resistance. Acquired resistance often involves mutation of drug targets or activation of compensatory pathways, making new targets in high demand. Physical contact of MM cells (MMC) with the bone microenvironment further protects MMC, via cell adhesion mediated-drug resistance (CAM-DR). Integrin α4β1 (Very Late Antigen 4; VLA4), mediates adhesion of MMC both to fibronectin in the matrix and to stromal cells expressing VCAM1, providing survival signals and protection from cell death upon drug treatment.Aiming to treat resistant cells, we proposed to utilize nanotechnology to safely deliver a drug whose target would not be lost in refractory MMC and to exploit, rather than bypassing, CAM-DR by introducing selective pressure for VLA4 overexpression then targeting it with drug-carrying nanoparticles (VLA4-NP). In particular, we hypothesized that, in combination treatments, VLA4-NP carrying a camptothecin prodrug (CPT-PD) would target drug-resistant MMC, reducing tumor burden and prolonging survival. Camptothecin (CPT) is a potent but poorly bioavailable topoisomerase 1 (TOP1) inhibitor. In vitro, CPT reduced proliferation and induced apoptosis in a panel of MM cell lines, including sub-lines resistant to other agents. RNAseq showed that MM cells from patients with relapsing or refractory MM had high levels of TOP1 expression. We modified CPT into an inactive lipase-sensitive prodrug (CPT-PD) that stabilizes the compound in micellar nanoparticles until enzymatically liberated in the MM cytosol (VLA4-CPT-PD-NP, 20 nm). In vitro and in vivo fluorescence tracking experiments showed that VLA4-NP binding increased in drug-resistant MM cells with minimal off-target delivery. In vitro, VLA4-CPT-PD-NP reduced MM cell proliferation and enhanced the effects of free drugs (melphalan, dexamethasone, lenalidomide). When MMC were co-cultured with primary bone marrow stroma from MM donors, MMC adhering to stroma survived free drug treatment, while VLA4-CPT-PD-NP had strong toxic effects on the myeloma cells and spared the stroma. In the KaLwRij/5TGM1 mouse model, melphalan resistant MMC are adherent to the bone. In these mice, uptake of fluorescently labeled VLA4-NP by MMC increased after melphalan treatment, while co-treatment with VLA4-CPT-PD-NP and free melphalan reduced tumor burden at multiple skeletal sites (p&amp;lt;0.01) and the M-protein levels by SPEP (p&amp;lt;0.01) relative to melphalan alone. Further, treatment with VLA4-CPT-PD and melphalan prolonged survival even after drug withdrawal (p&amp;lt;0.001), without added toxicity to the kidneys, liver or gut. This work provides proof of principle that CAM-DR can be exploited to safely deliver chemortherapeutics to resistant cells and effectively enhance treatment in myeloma. Citation Format: Francesca Fontana, Michael J. Scott, John S. Allen, Grace Cui, Xiaoxia Yang, Julie O'Neal, Steven Fletcher, Noriko Yanaba, Anne H. Schmieder, Michael P. Rettig, Julie Ritchey, Mark A. Fiala, Ravi Vij, Gregory M. Lanza. Nanotherapeutic targeting of drug resistant multiple myeloma cells through VLA4-contact facilitated drug-delivery of camptothecin prodrug [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1737.

Transcriptomic modifications of the thyroid gland upon exposure to phytosanitary-grade fipronil: Evidence for the activation of compensatory pathways

Fipronil is a phenylpyrazole insecticide used for the control of a variety of pest for domestic, veterinary and agricultural uses. Fipronil exposure is associated to thyroid disruption in the rat. It increases thyroid hormone (TH) hepatic clearance. The effect on thyroxine (T4) clearance is about four fold higher than the effect on T4 plasma concentrations suggesting that the thyroid gland might develop compensatory mechanisms. The aim of this study was to document the potential effects of fipronil treatment on the thyroid transcriptome together with its effects on TSH and TH blood levels under well characterized internal exposure to fipronil and its main metabolite fipronil sulfone.Fipronil (3 mg/kg/d by gavage for 14 days) clearance increased while its half-life decreased (about 10 fold) throughout treatment. Fipronil treatment in adult female rats significantly decreased total T4 and free triiodothyronine (T3) concentrations. Key genes related to thyroid hormone synthesis and/or cellular dynamic were modulated by fipronil exposure. RT-PCR confirmed that thyroglobulin gene expression was upregulated. A trend toward higher Na/I symporter expression was also noted, while sulfotransferase 1a1 gene expression was down-regulated. The expression of genes potentially involved in thyroid cell dynamic were upregulated (e.g. prostaglandin synthase 1, amphiregulin and Rhoa). Our results indicate that both pathways of TH synthesis and thyroid cell dynamics are transcriptional targets of fipronil and/or its main sulfone metabolite. The underlying mechanisms remain to be elucidated.