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

Supplementary Data from 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 (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.

Effects on cultured (human and mouse) immune cells

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient–derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC.Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo. These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression.Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted.Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell–based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153–64. ©2018 AACR.

Gene expression signatures used in Figure 1

Syngeneic mouse tumor efficacy, STAT3, and gene expression changes

Patient STAT reductions and reproducibility of replicates

Patient sample details

Supplementary Figure 1. Combination treatment with olaparib/AZD1775 is effective in select SCLC CDX models. Mice bearing CDX3 (a), CDX4 (b), CDX8 (c), or CDX8p (d) tumors were treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21 days. Each line depicts an individual relative tumor volume from all mice. e) CDX4 tumors were treated with vehicles (black) AZD1775 (red), topotecan (green), or the combination (blue). Topotecan was administered on days 1-3 and AZD1775 was administered on days 1-21. Supplementary Figure 2. CDX8p is more chemoresistant than CDX8. a) The clinical timeline for the patient that gave rise to CDX8/8p. Mice bearing CDX8 (b) or CDX8p (c) tumors were treated with vehicles (black) or cisplatin/etoposide (red). Individual relative tumor volumes are depicted. Supplementary Figure 3. Olaparib/AZD1775 is less effective after treatment with cisplatin/etoposide. Mice bearing CDX3 tumors were treated with cisplatin/etoposide on days 1-3 and allowed to recover for 3 weeks, at which time they were randomised and treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21days. The treatment periods are marked by orange lines. Individual relative tumor volumes are depicted for each mouse. Cohort sizes > 7. Supplementary Figure 4. AZD1775 and olaparib monotherapy activity in representative SCLC CDX models. CDX3 (a, b), CDX4 (c, d), CDX8 (e, f), or CDX8p (g, h) ex vivo cultures were treated with increasing concentrations of either AZD1775 (a, c, e, g) or olaparib (b, d, f, h) for 1 week and relative cell viability was assessed. One representative experiment of at least three is shown. Supplementary Figure 5. Representative immunohistochemical images for phospho-histone H3 (a), cleaved caspase 3 (b), phospho-CHK1 (c), and �H2AX (d) from CDX3 tumours treated with vehicle, AZD1775, olaparib, or the combination. Supplementary Figure 6. Lack of a BRCAness signature in olaparib-sensitive models. a) Heatmap of average log2 RPKM values are depicted for CDX3, CDX4, CDX8, and CDX8p following supervised clustering of models based on olaparib sensitivity. b) The number of synonymous (black) and nonsynonymous (white) mutations as determined by whole exome sequencing are graphed for CDX3, CD8, and CDX8p. The lack of a matched germline sample precluded somatic mutation calling in CDX4. c) Filtered RNAseq reads for PALB2 were loaded into the Broad Integrative Genomics Viewer and the E178* was highlighted in green for each read. Supplementary Figure 7. Published mechanisms of olaparib resistance. a) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. b) PARP1 RNA expression was assessed in tumor lysates from CDX3, CDX4, DX8, and CDX8p. Three representative tumors from each model were examined. c) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. Supplementary Figure 8. Treatment with AZD1775, olaparib, or the combination does not alter p21 expression. Duplicate tumor lysates taken 24 hours after indicated treatments were immunoblotted for indicated proteins. Supplementary Figure 9. Published mechanisms of AZD1775 resistance. a) SETD2 and PKMYT1 RNA expression was assessed in tumor lysates from CDX3, CDX4, CDX8, and CDX8p. Three representative tumors from each model were examined, and mean values are plotted against the cohort average maximal in vivo response to AZD1775. b) RRM2 levels were measured by immunoblotting lysates from CDX tumors harvested 2 hours after a single dose of vehicle (white), AZD1775 (red), olaparib (green), or the combination (blue). Samples were quantified and normalised to vinculin in each sample. Cohort sizes > 4 tumors per condition. Supplementary Table 1. Mass Specrometer and UPLC system parameters Supplementary Table 2. Optimization parameters for mass spectrometry analysis

Patient tumor gene expression data

Purpose:Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action.Experimental Design:Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen.Results:Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1.Conclusions:STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Cytokine, T, and NK cell changes in CT26 tumors

Gating and antibodies for flow cytometry and CyTOF