PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene’s reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

Table S2 shows data concerning the association between baseline characteristics and presence of BRCA reversion mutations in pretreatment circulating cell-free DNA

Supplementary Figure 6 - PDF file 112K, Impact of CO-1686, erlotinib and afatinib administration on mouse body-weight

A list of copy number alterations, rearrangements and short variants detected by Foundation Medicine NGS

Supplementary Tables, Figures and Video legends, Tables 2,3,5 and all Supplementary Figures. Supplementary Table 2. Confirmation of cis configuration of BRCA1 primary and secondary mutations in case 4 by colony PCR. Supplementary Table 3. IC50 (mircoM) values of the PARPi and platinum drugs in parental OVCAR8 cell line and OVCAR8 RAD51C KO clone, and the fold change in IC50 values. Supplementary Table 5. Sequences of primers used for site-directed mutagenesis. Supplementary Figure 1. Foundation Medicine NGS analysis of the 12 cases with archival tissue and/or pre-treatment and post-progression biopsies. Supplementary Figure 2. Sanger sequencing trace of the primary and secondary BRCA1 mutations in cis configuration in case 4 post-progression biopsy sample. Supplementary Figure 3. In vitro response to PARP inhibitor therapy and platinum agents in RAD51C deficient cell lines, with primary or secondary mutations in RAD51C. Supplementary Figure 4. RAD51 foci formation in geminin positive cells deficient for RAD51C, complemented with primary or secondary mutations in RAD51C. Supplementary Figure 5. Diagram of HR reporter assay. Supplementary Figure 6. RAD51C expression in MCF10A cells and in yeast. Supplementary Figure 7. Analysis of serial sections by direct PCR sequencing approach of a post-progression biopsy containing multiple secondary mutations in RAD51C. Supplementary Figure 8. Molecular Dynamics Modeling of WT RAD51D protein and RAD51D protein with secondary mutation c.770_776delinsA, p.S257_R259delinsK. Supplementary Figure 9. In vitro response to PARP inhibitor therapy and cisplatin in RAD51D deficient CHO cell line, with primary or secondary mutation in RAD51D. Supplementary Figure 10. Examination of the parental PEO4 cell line, PEO4 cells with the homozygous frameshift RAD51D mutation (c.762_763del, D254E*fs72) in the same exon as the primary mutation and PEO4 cells with the homozygous secondary RAD51D mutation (c.770_776delinsA, S257_R259delinsK). Supplementary Figure 11. Modeling of tumor clonal fractions in the post-progression biopsy sample with germline RAD51C mutation and multiple secondary mutations.

Supplementary Table 1 - PDF file 114K, Kinase selectivity profiling of CO-1686

PDF - 252K, Supplemental Table 1. Mass spectrometry on EGFR-L858R/T790M protein. Supplemental Table 2. EGFR modulation in A431, H1975 and HCC827 cells by compound 3. Supplemental Table 3. Kinase selectivity profile of compound 3, afatinib and WZ4002 at 1 ?M. Supplemental Table 4.Multi-dose level PK/PD study in H1975 tumor-bearing mice. Supplemental Figure 1. Signaling inhibition in EGFR wild-type (A431) and mutant EGFR NSCLC cells (H1975 and HCC827). Supplemental Figure 2. Prolonged duration of action on EGFR-L858R/T790M (A) and EGFR-DelE746-A750 (B) proteins. Supplemental Figure 3. Protein degradation (t1/2) of EGFR-L858R/T790M (A) and EGFR-DelE746-A750 (B) in H1975 and HCC827 cells, respectively. Supplemental Figure 4. Prolonged duration of action on EGFR-DelE746-A750 protein by erlotinib. Supplemental Figure 5. Time-dependent inhibition in EGFR mutant H1975 (A) and HCC827 (B) cells.

Molecular Dynamics Modeling of WT RAD51D monofilament interaction with dsDNA.

Supplementary Figure 5 - PDF file 134K, Schedule dependence of CO-1686 antitumor activity in NCI-H1975 mode

Supplementary Table 2 - PDF file 113K, Mutant EGFR cell panel profiling of CO-1686

Supplementary Figure 10 - PDF file 333K, Analysis of multiple EGFR-related signaling pathways in parental NCI-H1975 and COR resistant clones by Western blotting

Supplementary Figure 3 - PDF file 295K, Activity of CO-1686 against rare lung-cancer associated EGFR mutants

Figure S1 shows a consort diagram of patients with pretreatment and postprogression circulating cell-free DNA samples sequenced; Figure S2 shows a bar graph showing that a significantly lower level of the serum marker CA-125 was found at study enrollment in patients with no TP53 or BRCA mutations detected in the pretreatment circulating cell-free DNA; Figure S3 shows a scatter plot showing significant correlation between mutant allele frequency of primary somatic BRCA and TP53 mutations detected in pretreatment circulating cell-free DNA; Figure S4 shows a graph indicating the location of deleterious BRCA mutations where reversion mutations were detected in pretreatment and postprogression circulating cell-free DNA; Figure S5 shows a scatter plot showing a significant correlation of mutation allele frequency for the detected primary deleterious BRCA mutations between two independent next-generation sequencing-based circulating cell-free DNA assays; Figure S6 shows a swimlane graph showing the duration on rucaparib treatment of patients with or without BRCA reversion mutations detected in pretreatment circulating cell-free DNA; Figure S7 shows a graph from a linear regression analysis between the sum of mutation allele frequency of BRCA reversion mutations from pretreatment plasma and rucaparib progression-free survival; Figure S8 shows a bar graph of changes in BRCA reversion allele frequencies detected in pretreatment and postprogression circulating cell-free DNA from one patient.

Supplementary Figure 7 - PDF file 352K, H&E and immunohistochemical (Ki67) staining of tumors derived from EGFR mutant GEM models

Supplementary Figure 2 - PDF file 111K, CO-1686 inhibits cell growth in EGFR-mutant erlotinib-resistant cell lines

Supplementary Figure 11 - PDF file 255K, Analysis of the role of AXL in mediating CO-1686 resistance in COR cell clones.

Supplementary Materials and Methods - PDF file 179K, Supplemental materials and methods for Walter et al. manuscript

Supplementary Figure 4 - PDF file 103K, Pharmacokinetic analysis of CO-1686 in nu/nu mice

FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody. C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses. 177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86. In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+ T cells. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.

Copy number estimation by SNP array in the archival tumor tissue of the patient identified to have a germline RAD51C mutation (c.577C