Mechanisms of Resistance to PI3K Inhibitors in Cancer: Adaptive Responses, Drug Tolerance and Cellular Plasticity

Inhibidors de la via PI3K; Mecanismes de resistènciaInhibidores de la vía PI3K; Mecanismos de resistenciaPI3K pathway inhibitors; mechanisms of resistanceThe phosphatidylinositol-3-kinase (PI3K) pathway plays a central role in the regulation of several signalling cascades which regulate biological processes such as cellular growth, survival, proliferation, motility and angiogenesis. The hyperactivation of this pathway is linked to tumour progression and is one of the most common events in human cancers. Additionally, aberrant activation of the PI3K pathway has been demonstrated to limit the effectiveness of a number of anti-tumour agents paving the way for the development and implementation of PI3K inhibitors in the clinic. However, the overall effectiveness of these compounds has been greatly limited by inadequate target engagement due to reactivation of the pathway by compensatory mechanisms. Herein, we review the common adaptive responses that lead to reactivation of the PI3K pathway, therapy resistance and potential strategies to overcome these mechanisms of resistance. Furthermore, we highlight the potential role in changes in cellular plasticity and PI3K inhibitor resistance.The work was funded by a start-up grant provided by Curtin University

MiR-182-3p targets TRF2 and impairs tumor growth of triple-negative breast cancer

Target therapy; Telomeres; Triple-negative breast cancerTerapia dirigida; Telómeros; Cáncer de mama triple negativoTeràpia dirigida; Telòmers; Càncer de mama triple negatiuThe telomeric repeat-binding factor 2 (TRF2) is a telomere-capping protein that plays a key role in the maintenance of telomere structure and function. It is highly expressed in different cancer types, and it contributes to cancer progression. To date, anti-cancer strategies to target TRF2 remain a challenge. Here, we developed a miRNA-based approach to reduce TRF2 expression. By performing a high-throughput luciferase screening of 54 candidate miRNAs, we identified miR-182-3p as a specific and efficient post-transcriptional regulator of TRF2. Ectopic expression of miR-182-3p drastically reduced TRF2 protein levels in a panel of telomerase- or alternative lengthening of telomeres (ALT)-positive cancer cell lines. Moreover, miR-182-3p induced DNA damage at telomeric and pericentromeric sites, eventually leading to strong apoptosis activation. We also observed that treatment with lipid nanoparticles (LNPs) containing miR-182-3p impaired tumor growth in triple-negative breast cancer (TNBC) models, including patient-derived tumor xenografts (PDTXs), without affecting mouse survival or tissue function. Finally, LNPs-miR-182-3p were able to cross the blood–brain barrier and reduce intracranial tumors representing a possible therapeutic option for metastatic brain lesions.The research leading to these results has been funded by Italian Association for Cancer Research (AIRC # 21579) and Ministry of Health (CO-2019-12369662) to AB. This work was financially supported by Ministry of Health Ricerca Corrente 2022 and intramural grant-in-aid to EP. RD, LP and EP were supported by AIRC fellowships

Anti-tumoural activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumours

BRCA2; DNA damage responses; PyridostatinBRCA2; Respostes al dany de l'ADN; PiridostatinaBRCA2; Respuestas al daño del ADN; PiridotatinaThe cells with compromised BRCA1 or BRCA2 (BRCA1/2) function accumulate stalled replication forks, which leads to replication-associated DNA damage and genomic instability, a signature of BRCA1/2-mutated tumours. Targeted therapies against BRCA1/2-mutated tumours exploit this vulnerability by introducing additional DNA lesions. Because homologous recombination (HR) repair is abrogated in the absence of BRCA1 or BRCA2, these lesions are specifically lethal to tumour cells, but not to the healthy tissue. Ligands that bind and stabilise G-quadruplexes (G4s) have recently emerged as a class of compounds that selectively eliminate the cells and tumours lacking BRCA1 or BRCA2. Pyridostatin is a small molecule that binds G4s and is specifically toxic to BRCA1/2-deficient cells in vitro. However, its in vivo potential has not yet been evaluated. Here, we demonstrate that pyridostatin exhibits a high specific activity against BRCA1/2-deficient tumours, including patient-derived xenograft tumours that have acquired PARP inhibitor (PARPi) resistance. Mechanistically, we demonstrate that pyridostatin disrupts replication leading to DNA double-stranded breaks (DSBs) that can be repaired in the absence of BRCA1/2 by canonical non-homologous end joining (C-NHEJ). Consistent with this, chemical inhibitors of DNA-PKcs, a core component of C-NHEJ kinase activity, act synergistically with pyridostatin in eliminating BRCA1/2-deficient cells and tumours. Furthermore, we demonstrate that pyridostatin triggers cGAS/STING-dependent innate immune responses when BRCA1 or BRCA2 is abrogated. Paclitaxel, a drug routinely used in cancer chemotherapy, potentiates the in vivo toxicity of pyridostatin. Overall, our results demonstrate that pyridostatin is a compound suitable for further therapeutic development, alone or in combination with paclitaxel and DNA-PKcs inhibitors, for the benefit of cancer patients carrying BRCA1/2 mutations.Research in A.B. laboratory is supported by grants of Italian Association for Cancer Research (AIRC # 21579), Ministry of Health (CO 2019-12369662) and from IRCCS-Regina Elena Cancer Institute. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 722729 (F.J.G.). Research in M.T. laboratory is supported by Cancer Research UK (DRCPGM\100001), Medical Research Council and University of Oxford. A.R. received support from the UK Medical Research Council grant MC_PC_12006. V.S. received support from Instituto de Salud Carlos III (CPII19/00033, PI17/01080), GHD pink and the FERO Foundation

Alternative academic approaches for testing homologous recombination deficiency in ovarian cancer in the MITO16A/MaNGO-OV2 trial

Molecular testing; Ovarian cancerProves moleculars; Càncer d'ovarisPruebas moleculares; Cáncer de ovariosBackground The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. Patients and methods Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. Results The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen’s κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen’s κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. Conclusions Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation.This work was supported by funding from the AIRC [grant numbers IG 2016 – ID. 18921 and IG 2021 – ID. 25932 projects – P.I. SP and CO-2018-12367051 (Ministero della Salute) P.I SP]; Ricerca Corrente grant M2/7 from Ministero della Salute to DC, Ricerca Corrente from Ministero della Salute to SP. SM is supported by the Italian Association for Cancer Research [grant number IG-2017 n: IG19997]. MITO16A/MaNGO-OV2 trial was partially supported by Roche. AL is a recipient of a grant from the Asociación Española contra el Cáncer (AECC) [grant number INVES20095LLOP]. VS is a recipient of a grant from the Instituto de Salud Carlos III [grant number CPII19/00033] and a European grant for personalized medicine [grant number ERAPERMED 2019-215]. BP is a recipient of a grant from GOIRC. BP was supported by ESMO with a Clinical Translational Fellowship aid supported by Roche. Any views, opinions, findings, conclusions, or recommendations expressed in this material are those solely of the authors and do not necessarily reflect those of ESMO or Roche. NC has received funding from AstraZeneca (to the institution). FP has received funding from Roche, AstraZeneca, Pfizer, Merck Sharp & Dome, Bayer, Incyte, Taiho Oncology, Janssen Cilag, Exelixis, Aileron, and Daiichi Sankyo (grants to the institution for clinical trial activities)

AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kβ/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors

Cancer therapeutic resistance; Target identificationResistencia terapéutica contra el cáncer; Identificación de objetivosResistència terapèutica contra el càncer; Identificació d'objectiusThe PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kβi sensitivity while modulation of RRAGC, LAMTOR1, LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.This work was funded by the Wellcome Trust (WT206194) to KY and MJG and by AstraZeneca to KY, BRD, STB and JTL

Targeting mTOR to overcome resistance to hormone and CDK4/6 inhibitors in ER-positive breast cancer models

Cancer; Cell biologyCàncer; Biologia cel·lularCáncer; Biología celularResistance to therapy remains a major obstacle in cancer management. Although treatment with hormone and CDK4/6 inhibitors is successful in luminal breast cancer, resistance to these treatments is frequent, highlighting the need for novel therapeutic strategies to delay disease progression and improve patient survival. Here, we assessed the mechanisms of acquired resistance using T47D and MCF-7 tamoxifen- and palbociclib-resistant cell-line variants in culture and as xenografts, and patient-derived cells (PDCs) obtained from sensitive or resistant patient-derived xenografts (PDXs). In these models, we analyzed the effect of specific kinase inhibitors on survival, signaling and cellular aggressiveness. Our results revealed that mTOR inhibition is more effective than PI3K inhibition in overcoming resistance, irrespective of PIK3CA mutation status, by decreasing cell proliferation and tumor growth, as well as reducing cell migration and stemness. Moreover, a combination of mTOR and CDK4/6 inhibitors may prevent pathway reactivation downstream of PI3K, interfering with the survival of resistant cells and consequent tumor escape. In conclusion, we highlight the benefits of incorporating mTOR inhibitors into the current therapy in ER + breast cancer. This alternative therapeutic strategy not only enhances the antitumor response but may also delay the emergence of resistance and tumor recurrence.This work was supported by CONICET, ANPCYT (Grants PICT2509 & PICT0345), Instituto Nacional del Cáncer (Grants 2016 & 2018), Fundación Williams, Fundación Bunge & Born (Oster Grant) (Argentina); Instituto de Salud Carlos III (Grants PI20/00892 & CPII19/0033), Ministerio de Economía y Competitividad (FJCI-2015-25412) (Spain)

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors

Proteïnes supressores de tumors; Inhibidors de la quinasaProteínas supresoras de tumores; Inhibidores de la quinasaTumor suppressor proteins; Kinase inhibitorsCyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C–cyclin D–CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. Significance: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6–INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.The Chandarlapaty lab has received generous funding support for this research from the Cancer Couch Foundation, the Shen Family Fund, the Smith Fund for Cancer Research, the Breast Cancer Research Foundation, an NIH Cancer Center Support Grant (P30 CA008748), and NIH R01234361. Q. Li has received support from Translational Research Oncology Training Fellowship (MSKCC) made possible by the generous contribution of First Eagle Investment Management. V. Serra reports grants from the Susan G. Komen Foundation (CCR15330331) and Instituto de Salud Carlos III (CPII19/00033) during the conduct of the study and grants from Novartis, Genentech, and AstraZeneca outside the submitted work. The Chodera laboratory receives or has received funding from multiple sources, including the NIH and an NIH Cancer Center Support Grant (P30 CA008748), the National Science Foundation, the Parker Institute for Cancer Immunotherapy, Relay Therapeutics, Entasis Therapeutics, Silicon Therapeutics, EMD Serono (Merck KGaA), AstraZeneca, Vir Biotechnology, Bayer, XtalPi, Foresite Laboratories, the Molecular Sciences Software Institute, the Starr Cancer Consortium, the Open Force Field Consortium, Cycle for Survival, a Louis V. Gerstner Young Investigator Award, and the Sloan Kettering Institute. J. Guo acknowledges support from NIH grant R01 GM121505. J.D. Chodera acknowledges support from NIH grant P30 CA008748, NIH grant R01 GM121505, and NIH grant R01 GM132386. A complete funding history for the Chodera lab can be found at http://choderalab.org/funding, including complete funding information and grant numbers. The authors thank Dr. Marie Will and Madeline Dorso for helpful comments on the manuscript and Dr. Zhan Yao for helpful advice on the kinase assays

Analysis of matched primary and recurrent BRCA1/2 mutation-associated tumors identifies recurrence-specific drivers

Breast cancer; Cancer genetics; RNA sequencingCáncer de mama; Genética del cáncer; Secuenciación de ARNCàncer de mama; Genètica del càncer; Seqüenciació d'ARNRecurrence is a major cause of death among BRCA1/2 mutation carriers with breast (BrCa) and ovarian cancers (OvCa). Herein we perform multi-omic sequencing on 67 paired primary and recurrent BrCa and OvCa from 27 BRCA1/2 mutation carriers to identify potential recurrence-specific drivers. PARP1 amplifications are identified in recurrences (False Discovery Rate q = 0.05), and PARP1 is significantly overexpressed across primary BrCa and recurrent BrCa and OvCa, independent of amplification status. RNA sequencing analysis finds two BRCA2 isoforms, BRCA2-201/Long and BRCA2-001/Short, respectively predicted to be sensitive and insensitive to nonsense-mediated decay. BRCA2-001/Short is expressed more frequently in recurrences and associated with reduced overall survival in breast cancer (87 vs. 121 months; Hazard Ratio = 2.5 [1.18–5.5]). Loss of heterozygosity (LOH) status is discordant in 25% of patient’s primary and recurrent tumors, with switching between both LOH and lack of LOH found. Our study reveals multiple potential drivers of recurrent disease in BRCA1/2 mutation-associated cancer, improving our understanding of tumor evolution and suggesting potential biomarkers.This work was supported by the Tumor Tissue and Biospecimen Bank and the Next Generation Sequencing Core at the University of Pennsylvania (and in particular, we appreciated the help of Dr. Jonathan Schug). We would also like to thank Akoya Biosciences for their technical expertize regarding CODEX analyses. Lastly, the RNA sequencing pipeline used for this study was based on content from Dr. Dan Beiting’s DIY Transcriptomics class at the University of Pennsylvania (Spring 2021), for which we thank him as well. Figure 6 and Supplementary Fig. 15 were created using a licensed version of BioRender. This work was supported by the Basser Center for BRCA at the University of Pennsylvania (S.M.D., K.L.N.), the Gray Foundation Team Science Award (K.L.N.), the V Foundation for Cancer Research BRCA1/2 Convergence Team Award (K.L.N.), the Breast Cancer Research Foundation (S.M.D., K.L.N.) T32 HG009495 (A.K.), Miguel Servet Program (ISCIII) [CPII19/00033] (V.S.) and Asociación Española Contra el Cáncer (A.L.G.)

BRCA1 intronic Alu elements drive gene rearrangements and PARP inhibitor resistance

Resistència terapèutica contra el càncer; Càncer d'ovarisResistencia terapéutica al cáncer; Cáncer de ovariosCancer therapeutic resistance; Ovarian cancerBRCA1 mutant carcinomas are sensitive to PARP inhibitor (PARPi) therapy; however, resistance arises. BRCA1 BRCT domain mutant proteins do not fold correctly and are subject to proteasomal degradation, resulting in PARPi sensitivity. In this study, we show that cell lines and patient-derived tumors, with highly disruptive BRCT domain mutations, have readily detectable BRCA1 protein expression, and are able to proliferate in the presence of PARPi. Peptide analyses reveal that chemo-resistant cancers contain residues encoded by BRCA1 intron 15. Mechanistically, cancers with BRCT domain mutations harbor BRCA1 gene breakpoints within or adjacent to Alu elements in intron 15; producing partial gene duplications, inversions and translocations, and terminating transcription prior to the mutation-containing BRCT domain. BRCA1 BRCT domain-deficient protein isoforms avoid mutation-induced proteasomal degradation, support homology-dependent DNA repair, and promote PARPi resistance. Taken together, Alu-mediated BRCA1 gene rearrangements are responsible for generating hypomorphic proteins, and may represent a biomarker of PARPi resistance.This work was supported by the US National Institutes of Health (NIH) Grants P50 CA083638 and 5P30 CA006927, as well as R01CA214799, Susan Komen CCRCR17499048, and OC130212 Department of Defense to N.J., and a Pilot Award and Nested Teal Postdoctoral Scholar supported Y.W. (OC140040). We thank Drs. Judith Balmaña, Cristina Saura and Joaquin Arribas for providing materials. PDXs were established thanks to the GHD-Pink program, the FERO Foundation and the Catalan Agency AGAUR [2017 SGR 540]. V.S. is supported by the Instituto de Salud Carlos III (CP14/00228) and ALG by a PERIS fellowship from the Departament de Salut, Generalitat de Catalunya (SLT002/16/00477). Clovis Oncology supplied rucaparib. We are grateful to FCCC Genomics, Cell Culture and Cell Sorting facilities. We thank Hsin Yao Tang at the Wistar Proteomics facility for help with mass spectrometry

Clinical consequences of BRCA2 hypomorphism

Breast cancer; Cancer geneticsCáncer de mama; Genética del cáncerCàncer de mama; Genètica del càncerThe tumor suppressor FANCD1/BRCA2 is crucial for DNA homologous recombination repair (HRR). BRCA2 biallelic pathogenic variants result in a severe form of Fanconi anemia (FA) syndrome, whereas monoallelic pathogenic variants cause mainly hereditary breast and ovarian cancer predisposition. For decades, the co-occurrence in trans with a clearly pathogenic variant led to assume that the other allele was benign. However, here we show a patient with biallelic BRCA2 (c.1813dup and c.7796 A > G) diagnosed at age 33 with FA after a hypertoxic reaction to chemotherapy during breast cancer treatment. After DNA damage, patient cells displayed intermediate chromosome fragility, reduced survival, cell cycle defects, and significantly decreased RAD51 foci formation. With a newly developed cell-based flow cytometric assay, we measured single BRCA2 allele contributions to HRR, and found that expression of the missense allele in a BRCA2 KO cellular background partially recovered HRR activity. Our data suggest that a hypomorphic BRCA2 allele retaining 37–54% of normal HRR function can prevent FA clinical phenotype, but not the early onset of breast cancer and severe hypersensitivity to chemotherapy