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Supplementary Figure 3: NF2 deleterious mutations and sensitivity to lorlatinib (corresponding to main Figure 4).

Purpose:Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting.Experimental Design:Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance.Results:Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial–mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors.Conclusions:This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.

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Background Several selective FGFR inhibitors have been developed for the treatment of FGFR-driven urothelial cancer, in particular erdafitinib which received FDA and EMA approval. Molecular mechanisms responsible for resistance to FGFR inhibitors have not been elucidated in urothelial cancer. Understanding these resistance mechanisms is crucial for the development of novel agents and treatment strategies. Methods We identified patients with metastatic urothelial cancer harboring FGFR mutations or rearrangements, treated with selective FGFR inhibitors in the MATCH-R (NCT02517892), MOSCATO (NCT01566019), STING-UNLOCK (NCT04932525), and CTC studies at Gustave Roussy. Post-progression tissue biopsies and circulating tumor DNA (ctDNA) were analyzed with whole exome sequencing and Illumina or FoundationOne assays, respectively. We generated Ba/F3 models containing FGFR3::TACC3 transcript with the FGFR3 kinase domain mutations detected in post-progression samples and exposed them to eight FGFR inhibitors (readouts: cell viability assays and Western blots). We established patient-derived xenografts (PDX) and cell lines to assess resistance mechanisms and perform pharmacological studies. Results Twenty-one patients with post-progression samples (n = 13 both tissue and ctDNA, n = 5 ctDNA only, n = 3 tissue only) were included in our study. The molecular driver was identified as FGFR3 mutations/fusions in 19 cases, whereas one FGFR4 mutation and one FGFR2 rearrangement were found in two patients. Patients progressed to erdafitinib (n = 14), futibatinib (n = 4) or pemigatinib (n = 3). Thirteen, six, and two patients experienced response, stable disease, and progression, respectively. At progression, we detected single mutations in the FGFR kinase domain in six (29%) patients (FGFR3 N540K, V553L, V553M, V555L, V555M, E587Q; FGFR2 L551F) and multiple mutations in one (5%) case (FGFR3 N540K, V555L, L608V). Using Ba/F3 cells, we confirmed that the detected mutations conferred resistance to FGFR inhibitors. FGFR3 V553L, emerging in a patient treated with pemigatinib, was sensitive to erdafitinib and futibatinib. We detected alterations in the mTOR pathway in 11 (52%) patients (n = 5 TSC1/2, n = 5 PIK3CA, n = 1 NF2, n = 1 PTEN), with or without FGFR3 kinase domain mutations. In patient-derived models, erdafitinib combined with pictilisib was synergic in the presence of PIK3CA E545K, while erdafitinib-gefitinib combination overcame bypass resistance mediated by EGFR activation. Conclusions We detected a high frequency of FGFR kinase domain mutations at progression to selective FGFR inhibitors in urothelial cancer. Unlike FGFR2-driven cholangiocarcinoma, polyclonality of FGFR3 kinase domain mutations does not seem to be a hallmark of FGFR-driven urothelial cancer. The identification of off-target events and the preclinical validation sustain the potential clinical applicability of combinatorial treatment strategies. Citation Format: Francesco Facchinetti, Antoine Hollebecque, Floriane Braye, Damien Vasseur, Yoann Pradat, Rastislav Bahleda, Cédric Pobel, Ludovic Bigot, Olivier Déas, Juan-David Florez-Arango, Giorgia Guaitoli, Hayato Mizuta, David Combarel, Lambros Tselikas, Stefan Michiels, Sergey Nikolaev, Jean-Yves Scoazec, Santiago Ponce-Aix, Benjamin Besse, Ken A. Olaussen, Yohann Loriot, Luc Friboulet. Resistance to selective FGFR inhibitors in FGFR-driven urothelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3458.

Introduction: Advent of targeted therapies has deeply changed treatment of advanced oncogene-addicted non-small cell lung cancer (NSCLC), but development of resistance remains a main issue. Given the efficacy of next generation tyrosine kinase inhibitors (TKIs) against on-target mutations, their first-line administration could increase the relevance of off-target resistance mechanisms. Activating PIK3CA mutations and PTEN loss have been described as putative off-target resistance mechanisms across generations of EGFR TKIs. The role of these alterations in the development of resistance to ALK TKIs has been less described. MATCH-R trial (NCT02517892) is an ongoing prospective study whose objective is to understand mechanisms of acquired resistance to cancer therapies and develop strategies to overcome it. Materials and methods: We collected clinical and molecular data of patients (pts) with NSCLC enrolled in the molecular target group (MTG) of MATCH-R trial. We identified pts with EGFR mutations or ALK rearrangements who developed PIK3CA/PTEN alterations in tissue biopsy and/or circulating tumor DNA upon progression to first-line osimertinib/alectinib. We established patient-derived cell lines (PDCL) from the identified MATCH-R pts and we engineered commercial cell lines (CCL) harboring EGFR mutation (PC9) or ALK fusion (H3122) to constitutively express PIK3CA mutations and/or to have a permanent loss of PTEN. Cell viability assays and Western blot studies with different EGFR/ALK/PI3K/mTOR inhibitors were performed. Results: Of the 186 pts with advanced NSCLC included in the MTG of MATCH-R, 110 (59.1%) harbored EGFR mutations (EGFR+) and 27 (14.5%) ALK rearrangements (ALK+). Among them, 19 received first-line osimertinib and seven first-line alectinib. Five and two pts developed PIK3CA/PTEN alterations in the EGFR+ and ALK+ groups, respectively. In the EGFR+ group, PIK3CA E545K (n=2), R108H, N345K and R357Q mutations were detected. PTEN mutations (F437fs*5 and Y27C) were concomitant with E545K and R357Q mutations, respectively. In both ALK+ pts, a PIK3CA E545K mutation was identified, together with PTEN exon 5 splicing in one case. We confirmed on CCL that PIK3CA E545K mutation, alone or in combination with PTEN loss, confers resistance to second-/third-generation EGFR/ALK TKIs. PTEN loss alone had a moderate impact on TKIs sensitivity. Overcoming strategies combining EGFR/ALK TKIs with PIK3CA/mTOR inhibitors are being evaluated and will be disclosed at the meeting. Conclusions: Activating PI3KCA mutations and molecular events leading to PTEN loss are frequent events at resistance to first-line EGFR/ALK TKIs, and can be detected concomitantly. Functional assays confirmed that these alterations act as resistance mechanisms. These observations are of interest as PI3KCA/mTOR inhibitors may have a role in overcoming resistance. Citation Format: Giorgia Guaitoli, Francesco Facchinetti, Juan David Flórez-Arango, Floriane Braye, Hayato Mizuta, Santiago Ponce-Aix, Damien Vasseur, Ken André Olaussen, Stefan Michiels, Mihaela Aldea, Jordi Remon, Fabrice Barlesi, Benjamin Besse, David Planchard, Luc Friboulet. Alterations in PIK3CA/PTEN as resistance mechanisms in lung cancer patients progressing on first-line next generation EGFR/ALK tyrosine kinase inhibitors [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 3418.

Introduction: In the last 20 years, the advances in molecular oncology and cancer genetics allowed the identification of an increasing number of actionable oncogenic drivers and the development and clinical use of specific inhibitors. Despite these successes, it is now well established that tumour cells adapt and develop acquired resistance. It is crucial to understand these mechanisms of acquired resistance to develop overcoming therapeutic strategies. At Gustave Roussy, a prospective clinical trial MATCH-R (NCT02517892) is conducted to study the acquired resistance mechanisms and to find new therapeutic approaches for patients.. In parallel, of genetics analysis from patients biopsies, we develop Patient Derived Xenograft (PDX) to deepen our understanding of the resistance mechanism and to investigate new therapeutic approaches. Material and Method: Fresh tumor biopsy specimens were obtained prospectively from patients through a prospective single-institution clinical trial (MATCH-R, NCT02517892). Patient derived xenografts (PDX) in NOD Scid Gamma (NSG) mice as well as patient derived organoids from PDX (PDXO) and Patient derived cell lines were developed and characterized. Extensive molecular profiling including whole exome sequencing (WES), RNA sequencing (RNAseq) and immunohistochemistry were performed on human samples; PDX; Patient derived cells lines and PDXO. Results and Discussion: As of November 2022, 145 PDX models have been successfully obtained from 371 biopsies (global take rate of 39%). Our focus is the development of models from different cohorts: Androgen receptor inhibitors in castration-resistant prostate cancer, (18 PDX), ALK inhibitors in lung cancers (16 PDX including 3 post brigatinib, 7 post Lorlatinib and 6 Alectinib), EGFR inhibitors in lung cancers (33 PDX includind 24 post osimertinib), FGFR inhibitors in urothelial carcinoma and cholangiocarcinoma (29 PDX including 14 post erdafitinib, 4 post pemigatinib, 4 post futibatinib) and KRAS inhibitors in lung cancer and pancreatic cancers (20 PDX). The PDX models recapitulate the genetics, the phenotype and the pharmacology of the original biopsies. Novel mechanisms of resistance to tyrosine kinase inhibitors (TKI) in solid tumors were identified. Adaptive treatment with novel TKI or combinatorial strategies were evaluated to restore the sensitivity in PDX (readout: mean tumor growth). These results confirmed that PDX models are crucial to study the resistance mechanism and to develop new therapeutic strategies. Conclusion: Overall, the MATCH-R study provides a unique preclinical platform to identify resistance mechanisms to innovative therapies and to develop next generation therapeutic strategies. Citation Format: Ludovic Bigot, Catline Nobre, Francesco Facchinetti, Loic Poiraudeau, Floriane Braye, Jonathan Sabio, Naoual Mensourri, Olivier Deas, Claudio Nicotra, Maud Ngo-Camus, Lambros Tselikas, Jean Yves Scoazec, Karim Fizazi, Siantiago Ponce, Benjamin Besse, Luc Friboulet, Yohann Loriot. MatchR a preclinical platform of models resistant to innovative therapies. [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 4664.

Background Despite a primary benefit, resistance to the 3rd generation EGFR-Tyrosine Kinase Inhibitor (TKI) osimertinib invariably occurs. Several reports have recently highlighted the emergence of new oncogenic alterations as an actionable mechanism of resistance. However, the question of whether those alterations occur within a single tumor cell or in distinct tumor cell populations is still pending. Understanding those mechanisms at a cellular level is essential for a better comprehension of acquired resistance mechanisms and to identify new therapeutic opportunities. Methods This project studied tissues and patient derived cell lines from the ongoing prospective MATCH-R study (NCT0251782), in which patients with unresectable or metastatic cancer are included upon acquired resistance to targeted therapies or immunotherapy. Serial blood samples and tumor biopsies are collected at progression, for targeted NGS, WES and RNAseq, as well as PDX and patient-derived cell lines development. EGFR-mutated patients presenting a new driver alteration at osimertinib progression were identified. Single cell isolation and whole genome amplification were performed on corresponding frozen biopsies and derived cell lines. Cell lines were then exposed to specific inhibitors targeting the different pathways involved and functional analysis were performed to study the efficiency of combining different targeted therapies. Results Out of 466 patients included in MATCH-R study since June 2015, 110 patients presented EGFR-mutated adenocarcinoma. Among the 38 patients who experienced progression to 2nd line osimertinib, 5 patients were identified with a new oncogenic driver alteration, including STRN-ALK fusion (n=1), FGFR3-TACC3 fusion (n=1), BRAFV600E mutation (n=2), KIF-RET fusion (n=1). One patient progressing to 1st line osimertinib was identified with FGFR3-TACC3 fusion. All samples presented the persistence of EGFR activating mutation, i.e. EGFR exon 19 deletion (n=4) and exon 21 L858R mutation (n=2). After single cell isolation from tissue biopsies, PCR and targeted NGS allowed to highlight the co-existence of both drivers within single tumor cells for three patients. The remaining samples are still under investigation. Combination strategies with dual TKI are currently ongoing in order to restore sensitivity in cell lines (IC50 and Western Blots). Perspectives These data allow a better understanding of mechanisms underlying cell adaptation to EGFR-driven tumor inhibition. Combining targeted therapies represents a valuable therapeutic opportunity to overcome drug resistance in EGFR-mutated lung cancer. Updated results will be presented at the Meeting. Citation Format: Jeanne Chen, Floriane Braye, Francesco Facchinetti, Ludovic Lacroix, Jean-Yves Scoazec, Lambros Tselikas, David Planchard, Laura Mezquita, Anas Gazzah, Charles Naltet, Pernelle Lavaud, Aline Maillard, Stefan Michiels, Christophe Massard, Ken.A. Olaussen, Fabrice André, Gilles Vassal, Jean-Charles Soria, Benjamin Besse, Luc Friboulet. Characterization of multiple driver alterations in acquired resistance to osimertinib in EGFR-mutated lung cancer: implementation of single cell approaches [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 1867.

There are two isoforms of the anti-apoptotic protein BCL-2. BCL-2α (wild-type) is well characterised and is known predominantly for its role in apoptosis, BCL-2β lacks the C-terminal transmembrane (TM) domain and its function has not yet been characterised. The aim of this study was to quantify and determine the biological role of the BCL-2β isoform in melanoma. BCL-2α and BCL-2β isoforms were quantified at the mRNA and protein level in a cohort of 141 FFPE melanoma tumours using qPCR and multiple reaction monitoring (MRM) tandem mass spectrometry and compared to clinical parameters including age at diagnosis, gender, solar elastosis, BRAF/NRAS mutation, Breslow thickness, disease free survival and overall survival. The role of the BCL-2α and BCL-2β isoforms in apoptosis were investigated by quantifying transcript expression and apoptosis before and up to 72 hours after UVB irradiation (650J/m2) and cisplatin treatment (10ug/mL). siRNA knockdown targeted to each individual isoform transcript was used to verify the apoptotic response. Expression of the BCL-2β isoform in tumours was significantly associated with increased overall survival (686.4 weeks, 95% CI 462.5-910.3). BCL-2α response to UVB and cisplatin (i.e. downregulation prior to apoptosis) was the same in melanocyte and melanoma cell lines. However, BCL-2β response was varied across the melanoma cell lines, but was similar to that of BCL-2α in melanocytes. siRNA knock-down of BCL-2α resulted in increased apoptosis and cell death in melanoma cell lines, but knock-down of BCL-2β delayed onset of apoptosis, suggesting the BCL-2β is pro-apoptotic. Our current understanding of the role of BCL-2β is based on the concept that it lack the C-terminal transmembrane domain, and is incapable of localising to target organelles. It is currently thought that the isoform is of null function. However, these observations are based on studies of non-representative synthetic versions of BCL-2β. This is the first time the naturally transcribed version of the rare isoform has been studied. We have demonstrated herein that BCL-2β performs an apoptotic role, and that its regulation in melanoma may be a biomarker of better overall survival. Citation Format: Chloe Warren, Ricardo E. Vilain, Katie A. Ashton, Juhura G. Almazi, Stephen Braye, Pablo Moscato, Nikola A. Bowden. The rare BCL-2 isoform BCL-2β is associated with melanoma survival and the apoptotic response to UV and cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3559.

Global genome repair (GGR) is critical for recognizing DNA damage by the chemotherapy agent carboplatin, and triggering apoptotic signalling. XPC is a critical gene in the GGR pathway but in melanoma XPC is not induced in response to carboplatin resulting in extreme resistance. Despite low global methylation levels across the melanoma genome, there is evidence methylation plays a role in suppression of XPC. 5-aza-2’-deoxycytidine (decitabine), is a DNA methyltransferases inhibitor used as a chemotherapy agent that results in global loss of methylation and re-expression of silenced genes. We hypothesised that restoring expression of XPC in melanoma using decitabine could overcome resistance to carboplatin. To confirm low XPC in melanoma tumour tissue, transcript and protein expression from 196 advanced primary and metastatic melanomas was quantified and compared to normal tissue and other cancer types. Melanoma cell lines with low baseline XPC were treated with pharmacological doses of decitabine or carboplatin alone, or in sequential combination. XPC transcript, apoptosis, proliferation, senescence, global and CpG island shore demethylation was quantified. XPC transcript and protein was low in a proportion of melanoma tumours and expression correlated with overall survival. Treatment of melanoma cells with carboplatin alone did not significantly induce XPC expression or increase apoptosis in the majority of melanoma cell lines. Decitabine decreased global methylation (average 44.67% decrease) and increased XPC expression (0.9-3.0 fold increase). Hotspots of demethylation after decitabine treatment in the XPC CpG island shore were detected and could be responsible for the increase in XPC expression. When carboplatin was administered following decitabine, a greater XPC induction (1.5-7.6 fold increase) occurred with significantly increased levels of apoptosis (1.6-2.2 fold increase). This was coupled with a decreased proliferation and the presence of markers of senescence. Knocking down XPC expression with siRNA significantly reduced the effects of combination treatment. The outcomes of this study are evidence that silencing of XPC by methylation is a mechanism of carboplatin resistance in melanoma and sequential combination of decitabine followed by carboplatin may be used to restore GGR function and overcome resistance. Citation Format: Timothy Budden, Ryan J. Davey, Ricardo E. Vilain, Katie A. Ashton, Stephen Braye, Andre van der Westhuizen, Nikola A. Bowden. Global demethylation with decitabine increases DNA repair and sensitizes melanoma to carboplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4811.

Background: The BIG 1-98 trial, a large international Phase III study, evaluated letrozole (Let) for 5 years (n=1546) vs. Let for 2 years followed by tamoxifen (Tam) for 3 years (L→T; n=1540) and vs. Tam for 2 years followed by Let for 3 years (T→L; n=1548) as initial adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive breast cancer. Patients with HER2-positive disease did not receive trastuzumab. Efficacy results were presented in SABCS 2008 and showed that neither of the sequential therapy arms significantly improved disease-free survival (DFS) compared with letrozole alone. Centrally-assessed ER, PgR and HER2 have now been evaluated as potential predictive factors for treatment selection.Methods: Tumor blocks for 3975 of the 4634 (86%) patients on these three treatment arms were centrally collected and assessed by the IBCSG Central Pathology Laboratory. The tumors were evaluated for ER and PgR content by immunohistochemistry (IHC), and for HER2 immunoreactivity by IHC and by FISH for IHC 1+ or 2+ tumors. DFS according to centrally-assessed ER, PgR and HER2 status was analyzed using multivariable Cox modeling. STEPP methodology was used to explore patterns of DFS differences according to quantitative values of receptor levels (% staining cells).Results: Upon central review, 3885 tumors were confirmed to express ER and are the focus of this analysis. PgR expression did not predict differential treatment effects among the 3 treatment arms. With only 240 (6.2%) HER2-positive tumors, there was a suggestion of differential treatment benefit relative to HER2 status, in particular of a benefit of Let for 5 yrs over Tam→Let in HER2-positive tumors.Discussion: Given the caveat of a low prevalence of HER2-positive tumors and absence of HER2-directed therapy in the BIG 1-98 trial population, there is a suggestion that Let for 5 years may be a better option than a sequence of Tam and Let among women with HER2-positive tumors. Thus it may be better to initiate and continue treatment of endocrine-responsive, HER2+ tumors with Let rather than a sequence of Tam and Let. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 76.