Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance

Cáncer de mama y de ovario; Inhibición WEE1Càncer de mama i d'ovari; Inhibició WEE1Breast and ovarian cancer; WEE1 inhibitionPurpose: PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. Experimental Design: We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. Results: Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. Conclusions: Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.This work was supported by the Spanish Instituto de Salud Carlos III (ISCIII), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds (FIS PI17/01080 to V. Serra, PI12/02606 to J. Balmaña); European Research Area-NET, Transcan-2 (AC15/00063), Asociación Española contra el Cáncer (AECC; LABAE16020PORTT), the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR; 2017 SGR 540), La Marató TV3 (654/C/2019), and ERAPERMED2019–215 to V. Serra. We also acknowledge the GHD-Pink program, the FERO Foundation, and the Orozco Family for supporting this study (to V. Serra). V. Serra was supported by the Miguel Servet Program (ISCIII; CPII19/00033); M. Castroviejo-Bermejo and C. Cruz (AIOC15152806CRUZ) by AECC; A. Herencia-Ropero by Generalitat de Catalunya-PERIS (SLT017/20/000081); M. Palafox by Juan de la Cierva (FJCI-2015–25412); A. Lau by AECC and Generalitat de Catalunya-PERIS (INVES20095LLOP, SLT002/16/00477); A. Gris-Oliver by FI-AGAUR (2015 FI_B 01075). This work was supported by Breast Cancer Research Foundation (BCRF-19–08), Instituto de Salud Carlos III Project Reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (to J. Arribas). The xenograft program in the Caldas laboratory was supported by Cancer Research UK and also received funding from an EU H2020 Network of Excellence (EuroCAN). The RPPA facility is funded by NCI #CA16672

RAD51 as functional biomarker to select tumors for PARP inhibitor treatment

Los inhibidores de la enzima Poly (ADP-ribosa) polimerasa (PARPi) son efectivos en el tratamiento de cánceres que presentan defectos en la reparación del ADN por recombinación homóloga (HRR), incluyendo aquellos con mutaciones en BRCA1 y BRCA2 (BRCA1/2). Se han descrito distintos mecanismos de resistencia a PARPi en tumores con mutaciones germinales en BRCA1/2 (gBRCA), y existen otros tumores sin mutaciones en BRCA1/2 (no-BRCA) que responden a PARPi. Existe la necesidad de desarrollar un biomarcador robusto para una mejor selección de tumores deficientes en HRR y extender el uso de PARPi a nuevas indicaciones, así como estudiar terapias en combinación que mejoren la eficacia en la clínica. En este trabajo se evaluó la actividad del PARPi olaparib en xenoimplantes de tumores derivados de pacientes (PDX, patient derived tumor xenografts) con cáncer de mama u ovario, tanto gBRCA como no-gBRCA. Se estudiaron los mecanismos de resistencia y sensibilidad a PARPi in vivo, así como la utilidad de una inmunofluorescencia para detectar focos nucleares de RAD51 como biomarcador de HRR y respuesta a PARPi, tanto en PDXs como en muestras clínicas. Además, se investigó la actividad antitumoral del inhibidor de WEE1 AZD1775 y del inhibidor de ATM AZD0156 en monoterapia y combinación con PARPi. Se investigaron los mecanismos de acción de estas terapias utilizando distintos marcadores de estrés replicativo. Entre los modelos PDX gBRCA resistentes a PARPi no se encontraron mutaciones secundarias en BRCA1/2 pero sí la formación de focos nucleares de BRCA1, en concordancia con la expresión de proteínas BRCA1 hipomórficas. En tres PDX resistentes a PARPi se identificó la pérdida de 53BP1 y FAM35A como mecanismo de resistencia. La única característica común a todos los PDXs resistentes a PARPi, ya sea resistencia primaria o adquirida, fue la capacidad de formación de focos nucleares de la proteína RAD51. De acuerdo con estos resultados, la ausencia de focos de RAD51 se asoció con respuesta clínica a PARPi en muestras de pacientes. Cuando se estudiaron los mecanismos de sensibilidad a PARPi en la colección de PDXs no-gBRCA, se observó la presencia de hipermetilación del promotor de BRCA1 y alteraciones en otros genes relacionados con HRR en modelos sensibles a PARPi. Sin embargo, de nuevo la única característica común a todos los PDXs respondedores fue la ausencia de focos nucleares de RAD51. El ensayo de RAD51 se pudo realizar en muestras no tratadas y mostró ser altamente discriminativo entre sensibilidad y resistencia a PARPi, superando la capacidad predictiva del test genético myChoice® HRD de Myriad. En muestras clínicas de rutina procedentes de pacientes con síndrome de cáncer de mama y ovario hereditario, todos los tumores relacionados con mutaciones en PALB2 se clasificaron como deficientes en HRR. Finalmente, se demostró que la resistencia a la terapia con PARPi en tumores con alteraciones en BRCA1 puede revertirse combinando estos agentes con un inhibidor de WEE1 o de ATM, y en ambas estrategias se reportó una mayor inducción de estrés replicativo en PDX sensibles a la combinación. Los resultados de esta tesis permiten concluir que los tumores gBRCA logran la resistencia a PARPi mediante diferentes mecanismos que restauran HRR y puede detectarse por la formación de focos nucleares de RAD51. Este ensayo funcional permite identificar tumores no-BRCA sensibles a PARPi y representa un biomarcador prometedor para mejorar la selección de pacientes y ampliar la población candidata a recibir esta terapia. Los resultados también impulsan el desarrollo clínico de estrategias terapéuticas que combinen los PARPi con inhibidores de WEE1 y ATM, destacando la inducción de estrés replicativo como principal mecanismo de acción de estos fármacos.Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective anticancer drugs in cancers with defective homologous recombination DNA repair (HRR), including cancers with mutations in BRCA1 and BRCA2 (BRCA1/2), which also display enhanced sensitivity to DNA damaging chemotherapy such as platinum salts. Several mechanisms of PARPi resistance have been described in tumors with germline mutations in BRCA1/2 (gBRCA) and there are also other tumors with wild type BRCA1/2 (non-BRCA) that benefit from PARPi treatment. Therefore, there is a need to develop robust biomarkers to better select HRR- deficient tumors and extend the use of PARP inhibition in new indications, as well as identify PARPi-resistant tumors and study combination treatment options that enhance clinical efficacy and utility of PARPi. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from patients with breast or ovarian cancer, both with and without gBRCA mutation, exhibiting differential response to PARPi. We studied the in vivo mechanisms of PARPi resistance and sensitivity in these models and tested the formation of RAD51 nuclear foci by immunofluorescence as biomarker of HRR functionality and PARPi response in PDXs and routine clinical samples. We also tested the antitumor activity of the WEE1i AZD1775 and the ATMi AZD0156 as single agent and in combination with PARPi in PDXs. The measurement of replication stress biomarkers was assessed to study the mechanisms of action of these treatment strategies. Within the gBRCA PDXs panel, no BRCA1/2 secondary mutations were found in the PARPi resistant models. BRCA1 nuclear foci were detected in six out of ten PARPi-resistant PDXs, in keeping with expression of hypomorphic BRCA1 isoforms. Loss of 53BP1 and FAM35A were identified in three PDXs, one of which concomitantly expressed an hypomorphic BRCA1 protein. The common feature in all PDXs with primary or acquired PARPi resistance was the formation of RAD51 nuclear foci. Consistently, lack of RAD51 foci was always associated with clinical response to PARPi in patients treated with these agents. When studying the mechanisms of PARPi sensitivity in the non-gBRCA PDX cohort, BRCA1 promoter hypermethylation and alterations in HRR-related genes were found in PARPi- sensitive models. Again, the unique common feature in all PDXs that exhibited tumor regression upon PARPi treatment is the absence of RAD51 nuclear foci. The RAD51 assay could be performed in untreated samples and was highly discriminative of PARPi sensitivity versus PARPi resistance in different PDX cohorts and outperformed the Myriad’s myChoice® HRD genomic test. In routine clinical samples from patients with hereditary breast and ovarian cancer (HBOC) syndrome, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. In PDXs, PARPi resistance in BRCA1-altered tumors could be reverted upon combination of PARPi with WEE1 or ATM inhibitors and both combination strategies resulted in exacerbated induction of replication stress (RS) in combination- sensitive PDXs. With the results obtained in this thesis, it can be concluded that gBRCA tumors achieve PARPi resistance by several mechanisms that restore HRR function, all detected by the presence of RAD51 nuclear foci. This functional assay also enables the identification of PARPi-sensitive non-gBRCA tumors independently of the mechanisms of HRR-deficiency, thereby being a promising biomarker to better select patients for PARP inhibition and broaden the population who may benefit from this therapy. Our study also supports the clinical development of PARPi combinations such as those with WEE1 and ATM inhibitors and highlighted the induction of RS as the major mechanisms of action of these drugs

RAD51 as functional biomarker to select tumors for PARP inhibitor treatment

Los inhibidores de la enzima Poly (ADP-ribosa) polimerasa (PARPi) son efectivos en el tratamiento de cánceres que presentan defectos en la reparación del ADN por recombinación homóloga (HRR), incluyendo aquellos con mutaciones en BRCA1 y BRCA2 (BRCA1/2). Se han descrito distintos mecanismos de resistencia a PARPi en tumores con mutaciones germinales en BRCA1/2 (gBRCA), y existen otros tumores sin mutaciones en BRCA1/2 (no-BRCA) que responden a PARPi. Existe la necesidad de desarrollar un biomarcador robusto para una mejor selección de tumores deficientes en HRR y extender el uso de PARPi a nuevas indicaciones, así como estudiar terapias en combinación que mejoren la eficacia en la clínica. En este trabajo se evaluó la actividad del PARPi olaparib en xenoimplantes de tumores derivados de pacientes (PDX, patient derived tumor xenografts) con cáncer de mama u ovario, tanto gBRCA como no-gBRCA. Se estudiaron los mecanismos de resistencia y sensibilidad a PARPi in vivo, así como la utilidad de una inmunofluorescencia para detectar focos nucleares de RAD51 como biomarcador de HRR y respuesta a PARPi, tanto en PDXs como en muestras clínicas. Además, se investigó la actividad antitumoral del inhibidor de WEE1 AZD1775 y del inhibidor de ATM AZD0156 en monoterapia y combinación con PARPi. Se investigaron los mecanismos de acción de estas terapias utilizando distintos marcadores de estrés replicativo. Entre los modelos PDX gBRCA resistentes a PARPi no se encontraron mutaciones secundarias en BRCA1/2 pero sí la formación de focos nucleares de BRCA1, en concordancia con la expresión de proteínas BRCA1 hipomórficas. En tres PDX resistentes a PARPi se identificó la pérdida de 53BP1 y FAM35A como mecanismo de resistencia. La única característica común a todos los PDXs resistentes a PARPi, ya sea resistencia primaria o adquirida, fue la capacidad de formación de focos nucleares de la proteína RAD51. De acuerdo con estos resultados, la ausencia de focos de RAD51 se asoció con respuesta clínica a PARPi en muestras de pacientes. Cuando se estudiaron los mecanismos de sensibilidad a PARPi en la colección de PDXs no-gBRCA, se observó la presencia de hipermetilación del promotor de BRCA1 y alteraciones en otros genes relacionados con HRR en modelos sensibles a PARPi. Sin embargo, de nuevo la única característica común a todos los PDXs respondedores fue la ausencia de focos nucleares de RAD51. El ensayo de RAD51 se pudo realizar en muestras no tratadas y mostró ser altamente discriminativo entre sensibilidad y resistencia a PARPi, superando la capacidad predictiva del test genético myChoice® HRD de Myriad. En muestras clínicas de rutina procedentes de pacientes con síndrome de cáncer de mama y ovario hereditario, todos los tumores relacionados con mutaciones en PALB2 se clasificaron como deficientes en HRR. Finalmente, se demostró que la resistencia a la terapia con PARPi en tumores con alteraciones en BRCA1 puede revertirse combinando estos agentes con un inhibidor de WEE1 o de ATM, y en ambas estrategias se reportó una mayor inducción de estrés replicativo en PDX sensibles a la combinación. Los resultados de esta tesis permiten concluir que los tumores gBRCA logran la resistencia a PARPi mediante diferentes mecanismos que restauran HRR y puede detectarse por la formación de focos nucleares de RAD51. Este ensayo funcional permite identificar tumores no-BRCA sensibles a PARPi y representa un biomarcador prometedor para mejorar la selección de pacientes y ampliar la población candidata a recibir esta terapia. Los resultados también impulsan el desarrollo clínico de estrategias terapéuticas que combinen los PARPi con inhibidores de WEE1 y ATM, destacando la inducción de estrés replicativo como principal mecanismo de acción de estos fármacos.Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective anticancer drugs in cancers with defective homologous recombination DNA repair (HRR), including cancers with mutations in BRCA1 and BRCA2 (BRCA1/2), which also display enhanced sensitivity to DNA damaging chemotherapy such as platinum salts. Several mechanisms of PARPi resistance have been described in tumors with germline mutations in BRCA1/2 (gBRCA) and there are also other tumors with wild type BRCA1/2 (non-BRCA) that benefit from PARPi treatment. Therefore, there is a need to develop robust biomarkers to better select HRR- deficient tumors and extend the use of PARP inhibition in new indications, as well as identify PARPi-resistant tumors and study combination treatment options that enhance clinical efficacy and utility of PARPi. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from patients with breast or ovarian cancer, both with and without gBRCA mutation, exhibiting differential response to PARPi. We studied the in vivo mechanisms of PARPi resistance and sensitivity in these models and tested the formation of RAD51 nuclear foci by immunofluorescence as biomarker of HRR functionality and PARPi response in PDXs and routine clinical samples. We also tested the antitumor activity of the WEE1i AZD1775 and the ATMi AZD0156 as single agent and in combination with PARPi in PDXs. The measurement of replication stress biomarkers was assessed to study the mechanisms of action of these treatment strategies. Within the gBRCA PDXs panel, no BRCA1/2 secondary mutations were found in the PARPi resistant models. BRCA1 nuclear foci were detected in six out of ten PARPi-resistant PDXs, in keeping with expression of hypomorphic BRCA1 isoforms. Loss of 53BP1 and FAM35A were identified in three PDXs, one of which concomitantly expressed an hypomorphic BRCA1 protein. The common feature in all PDXs with primary or acquired PARPi resistance was the formation of RAD51 nuclear foci. Consistently, lack of RAD51 foci was always associated with clinical response to PARPi in patients treated with these agents. When studying the mechanisms of PARPi sensitivity in the non-gBRCA PDX cohort, BRCA1 promoter hypermethylation and alterations in HRR-related genes were found in PARPi- sensitive models. Again, the unique common feature in all PDXs that exhibited tumor regression upon PARPi treatment is the absence of RAD51 nuclear foci. The RAD51 assay could be performed in untreated samples and was highly discriminative of PARPi sensitivity versus PARPi resistance in different PDX cohorts and outperformed the Myriad’s myChoice® HRD genomic test. In routine clinical samples from patients with hereditary breast and ovarian cancer (HBOC) syndrome, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. In PDXs, PARPi resistance in BRCA1-altered tumors could be reverted upon combination of PARPi with WEE1 or ATM inhibitors and both combination strategies resulted in exacerbated induction of replication stress (RS) in combination- sensitive PDXs. With the results obtained in this thesis, it can be concluded that gBRCA tumors achieve PARPi resistance by several mechanisms that restore HRR function, all detected by the presence of RAD51 nuclear foci. This functional assay also enables the identification of PARPi-sensitive non-gBRCA tumors independently of the mechanisms of HRR-deficiency, thereby being a promising biomarker to better select patients for PARP inhibition and broaden the population who may benefit from this therapy. Our study also supports the clinical development of PARPi combinations such as those with WEE1 and ATM inhibitors and highlighted the induction of RS as the major mechanisms of action of these drugs

RAD51 as functional biomarker to select tumors for PARP inhibitor treatment

Los inhibidores de la enzima Poly (ADP-ribosa) polimerasa (PARPi) son efectivos en el tratamiento de cánceres que presentan defectos en la reparación del ADN por recombinación homóloga (HRR), incluyendo aquellos con mutaciones en BRCA1 y BRCA2 (BRCA1/2). Se han descrito distintos mecanismos de resistencia a PARPi en tumores con mutaciones germinales en BRCA1/2 (gBRCA), y existen otros tumores sin mutaciones en BRCA1/2 (no-BRCA) que responden a PARPi. Existe la necesidad de desarrollar un biomarcador robusto para una mejor selección de tumores deficientes en HRR y extender el uso de PARPi a nuevas indicaciones, así como estudiar terapias en combinación que mejoren la eficacia en la clínica. En este trabajo se evaluó la actividad del PARPi olaparib en xenoimplantes de tumores derivados de pacientes (PDX, patient derived tumor xenografts) con cáncer de mama u ovario, tanto gBRCA como no-gBRCA. Se estudiaron los mecanismos de resistencia y sensibilidad a PARPi in vivo, así como la utilidad de una inmunofluorescencia para detectar focos nucleares de RAD51 como biomarcador de HRR y respuesta a PARPi, tanto en PDXs como en muestras clínicas. Además, se investigó la actividad antitumoral del inhibidor de WEE1 AZD1775 y del inhibidor de ATM AZD0156 en monoterapia y combinación con PARPi. Se investigaron los mecanismos de acción de estas terapias utilizando distintos marcadores de estrés replicativo. Entre los modelos PDX gBRCA resistentes a PARPi no se encontraron mutaciones secundarias en BRCA1/2 pero sí la formación de focos nucleares de BRCA1, en concordancia con la expresión de proteínas BRCA1 hipomórficas. En tres PDX resistentes a PARPi se identificó la pérdida de 53BP1 y FAM35A como mecanismo de resistencia. La única característica común a todos los PDXs resistentes a PARPi, ya sea resistencia primaria o adquirida, fue la capacidad de formación de focos nucleares de la proteína RAD51. De acuerdo con estos resultados, la ausencia de focos de RAD51 se asoció con respuesta clínica a PARPi en muestras de pacientes. Cuando se estudiaron los mecanismos de sensibilidad a PARPi en la colección de PDXs no-gBRCA, se observó la presencia de hipermetilación del promotor de BRCA1 y alteraciones en otros genes relacionados con HRR en modelos sensibles a PARPi. Sin embargo, de nuevo la única característica común a todos los PDXs respondedores fue la ausencia de focos nucleares de RAD51. El ensayo de RAD51 se pudo realizar en muestras no tratadas y mostró ser altamente discriminativo entre sensibilidad y resistencia a PARPi, superando la capacidad predictiva del test genético myChoice® HRD de Myriad. En muestras clínicas de rutina procedentes de pacientes con síndrome de cáncer de mama y ovario hereditario, todos los tumores relacionados con mutaciones en PALB2 se clasificaron como deficientes en HRR. Finalmente, se demostró que la resistencia a la terapia con PARPi en tumores con alteraciones en BRCA1 puede revertirse combinando estos agentes con un inhibidor de WEE1 o de ATM, y en ambas estrategias se reportó una mayor inducción de estrés replicativo en PDX sensibles a la combinación. Los resultados de esta tesis permiten concluir que los tumores gBRCA logran la resistencia a PARPi mediante diferentes mecanismos que restauran HRR y puede detectarse por la formación de focos nucleares de RAD51. Este ensayo funcional permite identificar tumores no-BRCA sensibles a PARPi y representa un biomarcador prometedor para mejorar la selección de pacientes y ampliar la población candidata a recibir esta terapia. Los resultados también impulsan el desarrollo clínico de estrategias terapéuticas que combinen los PARPi con inhibidores de WEE1 y ATM, destacando la inducción de estrés replicativo como principal mecanismo de acción de estos fármacos.Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective anticancer drugs in cancers with defective homologous recombination DNA repair (HRR), including cancers with mutations in BRCA1 and BRCA2 (BRCA1/2), which also display enhanced sensitivity to DNA damaging chemotherapy such as platinum salts. Several mechanisms of PARPi resistance have been described in tumors with germline mutations in BRCA1/2 (gBRCA) and there are also other tumors with wild type BRCA1/2 (non-BRCA) that benefit from PARPi treatment. Therefore, there is a need to develop robust biomarkers to better select HRR- deficient tumors and extend the use of PARP inhibition in new indications, as well as identify PARPi-resistant tumors and study combination treatment options that enhance clinical efficacy and utility of PARPi. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from patients with breast or ovarian cancer, both with and without gBRCA mutation, exhibiting differential response to PARPi. We studied the in vivo mechanisms of PARPi resistance and sensitivity in these models and tested the formation of RAD51 nuclear foci by immunofluorescence as biomarker of HRR functionality and PARPi response in PDXs and routine clinical samples. We also tested the antitumor activity of the WEE1i AZD1775 and the ATMi AZD0156 as single agent and in combination with PARPi in PDXs. The measurement of replication stress biomarkers was assessed to study the mechanisms of action of these treatment strategies. Within the gBRCA PDXs panel, no BRCA1/2 secondary mutations were found in the PARPi resistant models. BRCA1 nuclear foci were detected in six out of ten PARPi-resistant PDXs, in keeping with expression of hypomorphic BRCA1 isoforms. Loss of 53BP1 and FAM35A were identified in three PDXs, one of which concomitantly expressed an hypomorphic BRCA1 protein. The common feature in all PDXs with primary or acquired PARPi resistance was the formation of RAD51 nuclear foci. Consistently, lack of RAD51 foci was always associated with clinical response to PARPi in patients treated with these agents. When studying the mechanisms of PARPi sensitivity in the non-gBRCA PDX cohort, BRCA1 promoter hypermethylation and alterations in HRR-related genes were found in PARPi- sensitive models. Again, the unique common feature in all PDXs that exhibited tumor regression upon PARPi treatment is the absence of RAD51 nuclear foci. The RAD51 assay could be performed in untreated samples and was highly discriminative of PARPi sensitivity versus PARPi resistance in different PDX cohorts and outperformed the Myriad's myChoice® HRD genomic test. In routine clinical samples from patients with hereditary breast and ovarian cancer (HBOC) syndrome, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. In PDXs, PARPi resistance in BRCA1-altered tumors could be reverted upon combination of PARPi with WEE1 or ATM inhibitors and both combination strategies resulted in exacerbated induction of replication stress (RS) in combination- sensitive PDXs. With the results obtained in this thesis, it can be concluded that gBRCA tumors achieve PARPi resistance by several mechanisms that restore HRR function, all detected by the presence of RAD51 nuclear foci. This functional assay also enables the identification of PARPi-sensitive non-gBRCA tumors independently of the mechanisms of HRR-deficiency, thereby being a promising biomarker to better select patients for PARP inhibition and broaden the population who may benefit from this therapy. Our study also supports the clinical development of PARPi combinations such as those with WEE1 and ATM inhibitors and highlighted the induction of RS as the major mechanisms of action of these drugs

Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance

PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. We analyzed breast and ovarian patientderived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi

Identification of a molecularly-defined subset of breast and ovarian cancer models that respond to WEE1 or ATR inhibition, overcoming PARP inhibitor resistance.

PURPOSE: PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR) deficient tumors and are used to treat breast, ovarian, pancreatic and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor combination demonstrated enhanced anti-tumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. EXPERIMENTAL DESIGN: We analyzed breast and ovarian patient-derived xenoimplant (PDX) models resistant to PARPi to quantify WEE1i and ATRi responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. RESULTS: While WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. CONCLUSION: Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi

A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2-related cancers. A test to identify additional HRR-deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2-related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi-sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2-related cancers

BRCA1(185delAG) tumors may acquire therapy resistance through expression of RING-less BRCA1

markdownabstractHeterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1 _185stop_ and Brca1 _5382stop_ alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1 _185delAG_ and BRCA1 _5382insC_. Both the Brca1185stop and Brca1 _5382stop_ mutations predisposed animals to mammary tumors, but Brca1 _185stop_ tumors responded markedly worse to HRD-targeted therapy than did Brca1 _5382stop_ tumors. Mice expressing Brca1 _185stop_ mutations also developed therapy resistance more rapidly than did mice expressing Brca1 _5382stop_. We determined that both murine Brca1 _185stop_ tumors and human BRCA1 _185delAG_ breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients