Search

Supplementary Tables

PARPi activates DNA damage responses and STING signaling pathway

Supplementary Figure 1. (A-B) Successful labeling of CellTrace Violet for M059K and H4 cells. M059K and H4 cells were labeled with a cell membrane dye CellTrace Violet and verified the success of labeling by FACS. (C-D) Cell viability of M059K and H4 following THIO treatment. M059K and H4 cells were treated with control medium or THIO at 5µM and then determined the cell viability percentage via detecting PSVue643 and PI by FACS. (E-F) Western blot results of time-course and dose-titration experiments in M059K and H4. For the time-course experiment, cells were treated with THIO at 5µM and proteins were tested at 0, 24, 48 and 72 hours. For dose-titration experiments, cells were treated with serious doses of THIO for 72 hours. TMZ functions as control. Actin serves as a loading control. Supplementary Figure 2. (A) Crystal violet staining images for acquired TMZ-resistant (TR) cell lines after treatment. (B) The heatmap of quantification results of the crystal violet images of TR cells. % relative to control. (C) Senescence assay for DBTRG-05MG, M059K and H4 cells following THIO treatment at 1 µM for 7 days, and then fixed and stained using Senescence β-Galactosidase Staining Kit and taken images by microscope. (D-F) Quantification results of senescence assay for DBTRG-05MG, M059K and H4 cells. 7 fields were counted for each group. (G-J) Neurosphere growth curves for T98G, U87MG, 13.0302 and TS-603 treated with or without THIO at 5 µM.

Supplementary Tables

Supplementary Figure S2

Supplementary Figure Legends

Purpose:To investigate the therapeutic role of a novel telomere-directed inhibitor, 6-thio-2′-deoxyguanosine (THIO) in gliomas both in vitro and in vivo.Experimental Design:A panel of human and mouse glioma cell lines was used to test therapeutic efficacy of THIO using cell viability assays, flow cytometric analyses, and immunofluorescence. Integrated analyses of RNA sequencing and reverse-phase protein array data revealed the potential antitumor mechanisms of THIO. Four patient-derived xenografts (PDX), two patient-derived organoids (PDO), and two xenografts of human glioma cell lines were used to further investigate the therapeutic efficacy of THIO.Results:THIO was effective in the majority of human and mouse glioma cell lines with no obvious toxicity against normal astrocytes. THIO as a monotherapy demonstrated efficacy in three glioma cell lines that had acquired resistance to temozolomide. In addition, THIO showed efficacy in four human glioma cell lines grown as neurospheres by inducing apoptotic cell death. Mechanistically, THIO induced telomeric DNA damage not only in glioma cell lines but also in PDX tumor specimens. Integrated computational analyses of transcriptomic and proteomic data indicated that THIO significantly inhibited cell invasion, stem cell, and proliferation pathways while triggering DNA damage and apoptosis. Importantly, THIO significantly decreased tumor proliferation in two PDO models and reduced the tumor size of a glioblastoma xenograft and a PDX model.Conclusions:The current study established the therapeutic role of THIO in primary and recurrent gliomas and revealed the acute induction of telomeric DNA damage as a primary antitumor mechanism of THIO in gliomas.

Supplementary Figure 1. (A-B) Successful labeling of CellTrace Violet for M059K and H4 cells. M059K and H4 cells were labeled with a cell membrane dye CellTrace Violet and verified the success of labeling by FACS. (C-D) Cell viability of M059K and H4 following THIO treatment. M059K and H4 cells were treated with control medium or THIO at 5µM and then determined the cell viability percentage via detecting PSVue643 and PI by FACS. (E-F) Western blot results of time-course and dose-titration experiments in M059K and H4. For the time-course experiment, cells were treated with THIO at 5µM and proteins were tested at 0, 24, 48 and 72 hours. For dose-titration experiments, cells were treated with serious doses of THIO for 72 hours. TMZ functions as control. Actin serves as a loading control.

Supplementary Figures Legends

Purpose:To investigate the antitumor activity of a mitochondrial-localized HSP90 inhibitor, Gamitrinib, in multiple glioma models, and to elucidate the antitumor mechanisms of Gamitrinib in gliomas.Experimental Design:A broad panel of primary and temozolomide (TMZ)-resistant human glioma cell lines were screened by cell viability assays, flow cytometry, and crystal violet assays to investigate the therapeutic efficacy of Gamitrinib. Seahorse assays were used to measure the mitochondrial respiration of glioma cells. Integrated analyses of RNA sequencing (RNAseq) and reverse phase protein array (RPPA) data were performed to reveal the potential antitumor mechanisms of Gamitrinib. Neurospheres, patient-derived organoids (PDO), cell line–derived xenografts (CDX), and patient-derived xenografts (PDX) models were generated to further evaluate the therapeutic efficacy of Gamitrinib.Results:Gamitrinib inhibited cell proliferation and induced cell apoptosis and death in 17 primary glioma cell lines, 6 TMZ-resistant glioma cell lines, 4 neurospheres, and 3 PDOs. Importantly, Gamitrinib significantly delayed the tumor growth and improved survival of mice in both CDX and PDX models in which tumors were either subcutaneously or intracranially implanted. Integrated computational analyses of RNAseq and RPPA data revealed that Gamitrinib exhibited its antitumor activity via (i) suppressing mitochondrial biogenesis, OXPHOS, and cell-cycle progression and (ii) activating the energy-sensing AMP-activated kinase, DNA damage, and stress response.Conclusions:These preclinical findings established the therapeutic role of Gamitrinib in gliomas and revealed the inhibition of mitochondrial biogenesis and tumor bioenergetics as the primary antitumor mechanisms in gliomas.

Purpose:On the basis of strong preclinical rationale, we sought to confirm recommended phase II dose (RP2D) for olaparib, a PARP inhibitor, combined with the AKT inhibitor capivasertib and assess molecular markers of response and resistance.Patients and Methods:We performed a safety lead-in followed by expansion in endometrial, triple-negative breast, ovarian, fallopian tube, or peritoneal cancer. Olaparib 300 mg orally twice daily and capivasertib orally twice daily on a 4-day on 3-day off schedule was evaluated. Two dose levels (DL) of capivasertib were planned: 400 mg (DL1) and 320 mg (DL-1). Patients underwent biopsies at baseline and 28 days.Results:A total of 38 patients were enrolled. Seven (18%) had germline BRCA1/2 mutations. The first 2 patients on DL1 experienced dose-limiting toxicities (DLT) of diarrhea and vomiting. No DLTs were observed on DL-1 (n = 6); therefore, DL1 was reexplored (n = 6) with no DLTs, confirming DL1 as RP2D. Most common treatment-related grade 3/4 adverse events were anemia (23.7%) and leukopenia (10.5%). Of 32 evaluable subjects, 6 (19%) had partial response (PR); PR rate was 44.4% in endometrial cancer. Seven (22%) additional patients had stable disease greater than 4 months. Tumor analysis demonstrated strong correlations between response and immune activity, cell-cycle alterations, and DNA damage response. Therapy resistance was associated with receptor tyrosine kinase and RAS-MAPK pathway activity, metabolism, and epigenetics.Conclusions:The combination of olaparib and capivasertib is associated to no serious adverse events and demonstrates durable activity in ovarian, endometrial, and breast cancers, with promising responses in endometrial cancer. Importantly, tumor samples acquired pre- and on-therapy can help predict patient benefit.

Supplementary materials

Supplementary Figure 3. (A) Relative initial telomere length of 17 glioma cell lines and NHA. (B) Correlation of IC50s of THIO and initial telomere length in 17 glioma cell lines. (C) Baseline relative expression of hTERT in 17 glioma cell lines and NHA. (D) RNAseq data of hTERT expression in 11 glioma cell lines obtained from CCLE. (E) Correlation of IC50s of THIO and baseline expression of hTERT in 17 glioma cell lines. (F) Correlation of IC50s of THIO and baseline expression of hTERT in 11 glioma cell lines from CCLE. (G) The heatmap of the enrichment scores of 5 telomere/telomerase-related gene signatures for 65 glioma cell lines from CCLE. (H-I) Relative telomere length of M059K and DBTRG-05MG cells following THIO treatment at 2.5 µM for 5 days. (J-K) Telomerase activity of M059K and DBTRG-05MG cells following THIO treatment at 5 µM for 72 hours. (L) Baseline telomerase activity of 10 glioma cell lines, including 5 cell lines that more sensitive to THIO and 5 cell lines that more resistant to THIO. (M) Correlation of baseline telomerase activity and cell viability (%) following THIO treatment. (N) Quantification of western blot of γH2AX expression in 8 glioma cell lines treated with THIO for 72 hours.

PARP inhibitors (PARPi) have shown remarkable therapeutic efficacy against BRCA1/2-mutant cancers through a synthetic lethal interaction. PARPi exert their therapeutic effects mainly through the blockade of ssDNA damage repair, which leads to the accumulation of toxic DNA double-strand breaks specifically in cancer cells with DNA repair deficiency (BCRAness), including those harboring BRCA1/2 mutations. Here we show that PARPi-mediated modulation of the immune response contributes to their therapeutic effects independently of BRCA1/2 mutations. PARPi promoted accumulation of cytosolic DNA fragments because of unresolved DNA lesions, which in turn activated the DNA-sensing cGAS–STING pathway and stimulated production of type I IFNs to induce antitumor immunity independent of BRCAness. These effects of PARPi were further enhanced by immune checkpoint blockade. Overall, these results provide a mechanistic rationale for using PARPi as immunomodulatory agents to harness the therapeutic efficacy of immune checkpoint blockade.Significance:This work uncovers the mechanism behind the clinical efficacy of PARPi in patients with both BRCA-wild-type and BRCA-mutant tumors and provides a rationale for combining PARPi with immunotherapy in patients with cancer.

Supplementary Data from Antitumor Activity of a Mitochondrial-Targeted HSP90 Inhibitor in Gliomas

Supplementary Figure 5. (A-C) The heatmap of proteins changed by THIO in M059K, LN229 and U87MG cells from RPPA data. (D-F) Western blot was performed to confirm the change of these proteins in H4 (more sensitive to THIO) (D), U87MG (less sensitive to THIO) (E), and U251MG (more resistant to THIO) (F) after treatment with THIO at 5 µM. Actin serves as a loading control. (G) Heatmap of pathways regulated by THIO in M059K, LN229 and U87MG cells.

Supplementary Figure 4. (A-D) The heatmap of significantly expressed gene in M059K, H4, D2224MG and DBTRG-05MG cells treated with or without THIO for 72 hours. |Log2 FC|>2.5 and p

Supplementary Figure 6. (A) Creation of primary patient-derived glioblastoma organoids. (B) Quantification of tumor weight of U87MG xenograft model. (C) The curve of body weight of mice of U87MG xenograft model during THIO treatment. (D) Quantification of tumor weight of GBM PDX model. (E) The curve of body weight of mice of GBM PDX model during THIO treatment. (F) The curve of body weight of mice of U251MG xenograft model during THIO treatment.

Supplementary Figure from Antitumor Activity of a Mitochondrial-Targeted HSP90 Inhibitor in Gliomas

Normal cells explore multiple states to survive stresses encountered during development and self-renewal as well as environmental stresses such as starvation, DNA damage, toxins or infection. Cancer cells co-opt normal stress mitigation pathways to survive stresses that accompany tumour initiation, progression, metastasis and immune evasion. Cancer therapies accentuate cancer cell stresses and invoke rapid non-genomic stress mitigation processes that maintain cell viability and thus represent key targetable resistance mechanisms. In this Review, we describe mechanisms by which tumour ecosystems, including cancer cells, immune cells and stroma, adapt to therapeutic stresses and describe three different approaches to exploit stress mitigation processes: (1) interdict stress mitigation to induce cell death; (2) increase stress to induce cellular catastrophe; and (3) exploit emergent vulnerabilities in cancer cells and cells of the tumour microenvironment. We review challenges associated with tumour heterogeneity, prioritizing actionable adaptive responses for optimal therapeutic outcomes, and development of an integrative framework to identify and target vulnerabilities that arise from adaptive responses and engagement of stress mitigation pathways. Finally, we discuss the need to monitor adaptive responses across multiple scales and translation of combination therapies designed to take advantage of adaptive responses and stress mitigation pathways to the clinic.

Purpose: To investigate the antitumor activity of a mitochondrial-localized HSP90 inhibitor, Gamitrinib, in multiple glioma models, and to elucidate the antitumor mechanisms of Gamitrinib in gliomas. Experimental Design: A broad panel of primary and temozolomide (TMZ)-resistant human glioma cell lines were screened by cell viability assays, flow cytometry, and crystal violet assays to investigate the therapeutic efficacy of Gamitrinib. Seahorse assays were used to measure the mitochondrial respiration of glioma cells. Integrated analyses of RNA sequencing (RNAseq) and reverse phase protein array (RPPA) data were performed to reveal the potential antitumor mechanisms of Gamitrinib. Neurospheres, patient-derived organoids (PDO), cell line–derived xenografts (CDX), and patient-derived xenografts (PDX) models were generated to further evaluate the therapeutic efficacy of Gamitrinib. Results: Gamitrinib inhibited cell proliferation and induced cell apoptosis and death in 17 primary glioma cell lines, 6 TMZ-resistant glioma cell lines, 4 neurospheres, and 3 PDOs. Importantly, Gamitrinib significantly delayed the tumor growth and improved survival of mice in both CDX and PDX models in which tumors were either subcutaneously or intracranially implanted. Integrated computational analyses of RNAseq and RPPA data revealed that Gamitrinib exhibited its antitumor activity via (i) suppressing mitochondrial biogenesis, OXPHOS, and cell-cycle progression and (ii) activating the energy-sensing AMP-activated kinase, DNA damage, and stress response. Conclusions: These preclinical findings established the therapeutic role of Gamitrinib in gliomas and revealed the inhibition of mitochondrial biogenesis and tumor bioenergetics as the primary antitumor mechanisms in gliomas.

INTRODUCTION: Drug resistance is the main barrier to achieving cancer cures with medical therapy. Cancer drug resistance occurs, in part, due to adaptation of the tumor and microenvironment to therapeutic stress at a proteomic level. Reverse-phase protein arrays (RPPA) are well-suited to proteomic analysis of drug resistance due to high sample throughput, sensitive detection of phosphoproteins, and validation for a large number of critical cellular pathways. AREAS COVERED: This review summarizes contributions of RPPA to understanding and combating drug resistance. In particular, contributions of RPPA to understanding resistance to PARP inhibitors, BRAF inhibitors, immune checkpoint inhibitors, and breast cancer investigational therapies are discussed. Articles reviewed were identified by MEDLINE, Scopus, and Cochrane search for keywords “proteomics,” “reverse-phase protein array,” “drug resistance,” “PARP inhibitor,” “BRAF inhibitor,” “immune checkpoint inhibitor,” and “I-SPY” spanning October 1 1960 – October 1 2021. EXPERT OPINION: Precision oncology has thus far failed to convert the armament of targeted therapies into durable responses for most patients, highlighting that genetic sequencing alone is insufficient to guide therapy selection and overcome drug resistance. Combined genomic and proteomic analyses paired with creative drug combinations and dosing strategies hold promise for maturing precision oncology into an era of improved patient outcomes.

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.

We investigated the tumor microenvironment (TME) in estrogen receptor positive (ER+) primary breast cancers from stage I-III patients treated with estrogen deprivation (ED, induced with letrozole) for 10-21 days. We used primary breast cancer biopsies, collected from 198 patients, to assess tumor-infiltrating lymphocytes (TILs), transcriptomic profiles, and TME composition. Diagnostic biopsy and surgical on-treatment tumor specimens were collected; Ki67, ERα, PR, and HER2 expression were measured using automated quantitative analysis (AQUA). Tumors were categorized as estrogen deprivation-sensitive (ED-S) or -resistant (ED-R) based on the natural log (ln) of the Ki67 score (ln ≤1.0 or ≤2.7% Ki67+ cells vs. ln ≥2.0 or ≥7.4%, respectively) in the on-treatment (surgical) biopsy. Scoring of stromal (s)TILs, using hematoxylin and eosin staining of on-treatment specimens, revealed that sTILs were elevated in ED-R compared to ED-S tumors (p Citation Format: Fabiana Napolitano, Dhivya R. Sudhan, Yunguan Wang, Paula I. González-Ericsson, Luigi Formisano, Kyung-Min Lee, Chang-Ching Lin, María Rosario Chica-Parrado, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Justin M. Balko, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy [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 3445.

The treatment of metastatic melanoma has been revolutionized with the introduction of immune checkpoint blockade (ICB) therapies in the last decade. Despite durable responses exhibited by a subgroup of patients, most of patients do not respond to ICB therapies and the vast majority (60%-90%) of patients experience disease progression within five years. Molecular mechanisms of primary and acquired resistance to ICB are still widely unknown, which warrants further mechanistic investigation. Towards that goal, we used a holistic approach driven by integrated genomic tools to molecularly profile 35 longitudinal tumor specimens at pre-, on-, and post-treatment time points which were derived from seven patients with metastatic melanoma who progressed on sequential ICB therapies, including three responders and four non-responders. We integrated analyses of data generated from RNA sequencing, Whole-exome Sequencing (WES), NanoString nCounter vantage 3D, Cyclic Immunofluorescence (CyCIF), NanoString Digital Spatial Profiling (DSP) and Multiplex Immunohistochemistry (mIHC) platforms to elucidate evolutionary trajectories that occurred in both the tumor and the tumor immune microenvironment (TiME) that may have orchestrated response and resistance to ICB therapies. Simultaneous analyses conducted on genomic, transcriptomic and proteomic levels identified the re-activation of the MAPK pathway as a potential mechanism of resistance to ICB therapies. Spatially-resolved, image-based immune monitoring analysis at single cell resolution revealed that response to ICB is associated with infiltration of immune cells accompanied by induced activities of myeloid, T cells and macrophages in the TiME. In summary, integrated analyses allow us not only to reveal the dynamic co-revolution of both tumor and immune cells in TiME following sequential ICB therapies but also to provide a comprehensive perspective to molecular mechanisms of response and resistance to ICB therapies. A deeper understanding and elucidation of the evolution of mechanisms of response and resistance to ICB therapies will point us to generate hypothesis-driven, potential salvage therapies to overcome resistance to ICB therapies. Citation Format: Shiyou Wei, Jinho Lee, Marilyne Labrie, Courtney Betts, Lunxu Liu, Lisa Coussens, Meenhard Herlyn, Genevieve Boland, Gordon Mills, Gao Zhang. Spatiotemporal analysis of metastatic melanoma reveals mechanisms of resistance to immune checkpoint blockade [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 2276.

Background: Clear cell ovarian carcinoma (CCOC) is characterized by a distinct histologic and molecular profile, and associated with very poor responses to standard treatment consisting of surgery and carboplatin:taxol chemotherapy. CCOC is chemo-resistant at the time of diagnosis and response to chemotherapy in the recurrent setting is less than 10%. Literature suggests a potential role for immune checkpoint inhibitors (ICI). However, only a subgroup of patients (20%) responds to ICI and little is known about the mechanisms of response and resistance to therapy. We postulate that a better understanding of CCOC tumor microenvironment (TME) could help predict patients’ response to ICI. Objective: The aim of this study is to investigate the relationship between TME immune infiltrate and clinical/outcome in 22 CCOC cases and identify subsets of CCOC who may benefit from immunotherapy. Material & Methods: We characterized the immune landscape of 11 early and 11 advanced CCOC through a multiplex IHC Discovery Platform. Spatial single-cell proteomics analyses (cyclic-IF) and spatially-resolved RNAseq in 10 CCOC cases using an OC tissue microarray (TMA) were performed. Results were corelated with clinical and treatment outcome. Results: The percent of CD8, CD4, CD20 B cells Tregs, PD1, PDL1, monocytes and M2 monocytes and myeloid cells was significantly higher in advanced than early-stage cancers. Recurrent cancers were more immunosuppressive than cases with no recurrence. Tumor infiltrate was dense in 4 cases. Four patients with early-stage disease had a high number of CD8 naïve cells and experienced no recurrence. TME analysis of single case of advanced CCOC (before and after chemotherapy), revealed that hot tumor changed to cold tumor, suggesting the resistance to treatment. Cyclic IF analyses identified 3 tumor phenotype groups in a subset of 10 CCOC present on the OC TMA. The majority of CCOC had high expression of CCNE and high PI3K-AKT-mTor activity, low expression of hormone receptors (AR, ERa, PRg) and low cell cycle activity. Some tumors were also high for HER2. The stromal compartment was enriched in collagen VI, aSMA and PDGFR. The immune monitoring of several of those samples also revealed an immunosuppressive microenvironment, including the presence of M2 macrophages and expression of immune checkpoint proteins PD-L1 and B7-H4. Conclusion: Our study shows that various phenotypes of CCOC are defined by the cancer cell profiles and TME content. Importantly, a strong immunosuppressive microenvironment was detected in many samples, suggesting a potential response to ICI. Furthermore, we detected the expression of several therapeutic targets (MAPK, CCNE, PI3K-AKT-mTOR, HSP90, HER2), including oncogenic signaling pathways (RTK, MAPK). Different tumor phenotypes identified across our CCOC samples suggest that clear cell carcinoma could be subclassified into subtypes that should be treated differently. Citation Format: Tanja Pejovic, Sonali Joshi, Shawn Campbell, Dhanir Tailor, Joanna Pucilowska, Benjamin Tate, Pierre-Valérien Abate, Korina Mouzakitis, Marilyne Labrie, Elizabeth Munro, Jenna Emerson, Sanjay V. Malhotra. Study of tumor microenvironment of ovarian clear cell carcinoma [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 79.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated activity across a broad spectrum of molecular backgrounds and tumor types, with the greatest activity observed in patients with aberrations in the homologous recombination DNA damage repair pathway. Despite remarkable responses in a subset of patients, the response is usually modest and transient due to the almost inevitable emergence of resistance. Tumors develop resistance through rapid adaptation to the effects of PARPi as well as by generation or selection of genomic aberration. Although adaptive responses results in drug resistance, it also induces therapeutic vulnerabilities that could be exploited with rational combination therapies. To fulfill this role, we established the combinatorial adaptive response therapy (CART) platform by performing reverse-phase protein arrays to characterize adaptive responses, and develop rational combination therapies. Our series of studies strongly support the efficacy of this strategy, wherein targeting the emerging adaptive responses to PARPi with MEK/ERK inhibitors, WEE1/ATR inhibition (inhibitors of S-phase and G2 DNA damage checkpoint), and PI3K/AKT/mTOR inhibition, and showed promising anti-tumor activity in various preclinical models. Importantly, this approach has been proven highly efficient, and several combinational therapies developed from the CART platform are being evaluated in ongoing clinical trials (NCT03801369, NCT03586661, NCT03162627, NCT03544125, NCT02659241, NCT02208375, NCT02316834, and NCT03637491).

Purpose: To investigate the therapeutic role of a novel telomere-directed inhibitor, 6-thio-2′-deoxyguanosine (THIO) in gliomas both in vitro and in vivo. Experimental Design: A panel of human and mouse glioma cell lines was used to test therapeutic efficacy of THIO using cell viability assays, flow cytometric analyses, and immunofluorescence. Integrated analyses of RNA sequencing and reverse-phase protein array data revealed the potential antitumor mechanisms of THIO. Four patient-derived xenografts (PDX), two patient-derived organoids (PDO), and two xenografts of human glioma cell lines were used to further investigate the therapeutic efficacy of THIO. Results: THIO was effective in the majority of human and mouse glioma cell lines with no obvious toxicity against normal astrocytes. THIO as a monotherapy demonstrated efficacy in three glioma cell lines that had acquired resistance to temozolomide. In addition, THIO showed efficacy in four human glioma cell lines grown as neurospheres by inducing apoptotic cell death. Mechanistically, THIO induced telomeric DNA damage not only in glioma cell lines but also in PDX tumor specimens. Integrated computational analyses of transcriptomic and proteomic data indicated that THIO significantly inhibited cell invasion, stem cell, and proliferation pathways while triggering DNA damage and apoptosis. Importantly, THIO significantly decreased tumor proliferation in two PDO models and reduced the tumor size of a glioblastoma xenograft and a PDX model. Conclusions: The current study established the therapeutic role of THIO in primary and recurrent gliomas and revealed the acute induction of telomeric DNA damage as a primary antitumor mechanism of THIO in gliomas.

// Marilyne Labrie 1 , Tae-Beom Kim 2 , Zhenlin Ju 2 , Sanghoon Lee 3 , Wei Zhao 3 , Yong Fang 1 , Yiling Lu 3 , Ken Chen 2 , Pedro Ramirez 4 , Michael Frumovitz 4 , Larissa Meyer 4 , Nicole D. Fleming 4 , Anil K. Sood 4 , Robert L. Coleman 4 , Gordon B. Mills 1 , 3 and Shannon N. Westin 4 1 Knight Cancer Institute and Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA 2 Department of Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA 3 Department of Systems Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA 4 Department of Gynecologic Oncology and Reproductive Medicine, University of Texas, MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Shannon N. Westin, email: swestin@mdanderson.org Keywords: poly (ADP-ribose) polymerase inhibitor; combination therapy; adaptive response; ovarian cancer; targeted therapy Received: March 12, 2019 Accepted: May 02, 2019 Published: May 28, 2019 ABSTRACT Poly (ADP-ribose) polymerase inhibitor (PARPi)-based combination therapies are demonstrating efficacy in patients, however, identifying the right combination for the right patient remains a critical challenge. Thus, it is urgent to develop approaches able to identify patients likely to benefit from specific combination therapies. Several groups, including ours, have demonstrated that targeting adaptive responses induced by PARPi increases depth and duration of response. In this study, we instituted a talazoparib (PARPi) monotherapy window of opportunity trial to identify informative adaptive responses in high grade serous ovarian cancer patients (HGSOC). Patients were treated for 7 to 14 days with PARPi monotherapy prior to surgery with tissue samples from multiple sites being collected pre- and post-treatment in each patient. Analysis of these samples demonstrated that individual patients displayed different adaptive responses with limited interlesional heterogeneity. Ability of combination therapies designed to interdict adaptive responses to decrease viability was validated using model systems. Thus, assessment of adaptive responses to PARPi provides an opportunity for patient-specific selection of combination therapies designed to interdict patient-specific adaptive responses to maximize patient benefit.

PARP inhibitors (PARPi) have shown remarkable therapeutic efficacy against BRCA1/2-mutant cancers through a synthetic lethal interaction. PARPi exert their therapeutic effects mainly through the blockade of ssDNA damage repair, which leads to the accumulation of toxic DNA double-strand breaks specifically in cancer cells with DNA repair deficiency (BCRAness), including those harboring BRCA1/2 mutations. Here we show that PARPi-mediated modulation of the immune response contributes to their therapeutic effects independently of BRCA1/2 mutations. PARPi promoted accumulation of cytosolic DNA fragments because of unresolved DNA lesions, which in turn activated the DNA-sensing cGAS–STING pathway and stimulated production of type I IFNs to induce antitumor immunity independent of BRCAness. These effects of PARPi were further enhanced by immune checkpoint blockade. Overall, these results provide a mechanistic rationale for using PARPi as immunomodulatory agents to harness the therapeutic efficacy of immune checkpoint blockade. Significance: This work uncovers the mechanism behind the clinical efficacy of PARPi in patients with both BRCA-wild-type and BRCA-mutant tumors and provides a rationale for combining PARPi with immunotherapy in patients with cancer.

Image-based cell phenotyping relies on quantitative measurements as encoded representations of cells; however, defining suitable representations that capture complex imaging features is challenged by the lack of robust methods to segment cells, identify subcellular compartments, and extract relevant features. Variational autoencoder (VAE) approaches produce encouraging results by mapping an image to a representative descriptor, and outperform classical hand-crafted features for morphology, intensity, and texture at differentiating data. Although VAEs show promising results for capturing morphological and organizational features in tissue, single cell image analyses based on VAEs often fail to identify biologically informative features due to uninformative technical variation. Herein, we propose a multi-encoder VAE (ME-VAE) in single cell image analysis using transformed images as a self-supervised signal to extract transform-invariant biologically meaningful features, including emergent features not obvious from prior knowledge. We show that the proposed architecture improves analysis by making distinct cell populations more separable compared to traditional VAEs and intensity measurements by enhancing phenotypic differences between cells and by improving correlations to other analytic modalities.

BackgroundCombining poly (ADP-ribose) polymerase (PARP) with phosphatidylinositol-3-kinase (PI3K) pathway inhibitors is supported by strong preclinical rationale. We sought to assess safety and determine a recommended phase 2 dose (RP2D) for PARP inhibitor olaparib combined with the AKT inhibitor, capivasertib, and evaluate molecular markers of response and resistance.MethodsAs part of a larger phase 1b trial, we performed a safety lead in of olaparib and capivasertib followed by expansion (n=24) in endometrial, triple negative breast, ovarian, fallopian tube, or peritoneal cancer. Olaparib 300mg orally twice daily and capivasertib orally twice daily on a four day on three day off schedule was evaluated. Two dose levels (DL) were planned: capivasertib 400mg (DL1); capivasertib 320mg (DL-1). Patients underwent biopsies at baseline and after 28 days.Findings38 patients were enrolled. 7 (18%) patients had known germlineBRCA1/2mutations. The first two patients on DL1 experienced dose limiting toxicities (DLTs) of diarrhea and vomiting in absence of maximum supportive care. No DLTs were observed on DL-1 (n=6), therefore, DL1 was re-explored (n=6) with no DLTs, confirming this as RP2D. Most common treatment-related grade 3 or 4 adverse events were anemia (23.7%) and leukopenia (10.5%).Of 32 subjects evaluable for response, 6 (19%) had partial response (PR) with a PR rate of 44.4% in endometrial cancer. Seven (22%) additional patients had stable disease greater than 4 months. Tumor analysis demonstrated strong correlation between response and immune activity, as well as alterations in cell cycle and DNA damage response genes. Therapy resistance was associated with receptor tyrosine kinase (RTK) and RAS-MAPK pathway activity, as well as metabolism and epigenetics.InterpretationThe combination of olaparib and capivasertib is well tolerated and demonstrates evidence of durable activity in women’s cancers, with particularly promising response in endometrial cancer. Importantly, tumor samples acquired pre and on-therapy can help predict patient benefit.FundingAstraZeneca, MDACC Moonshots Program, MDACC Support Grant CA016672 NCI SPOREs in Ovarian (CA217685) and Uterine (CA098258) Cancer and a kind gift from the Miriam and Sheldon Medical Research Foundation. AZD5363 was discovered by AstraZeneca subsequent to a collaboration with Astex Therapeutics (and its collaboration with the Institute of Cancer Research and Cancer Research Technology Limited).

Molecular heterogeneity in metastatic breast cancer presents multiple clinical challenges in accurately characterizing and treating the disease. Current diagnostic approaches offer limited ability to assess heterogeneity that exists among multiple metastatic lesions throughout the treatment course. We developed a precision oncology platform that combines serial biopsies, multi-omic analysis, longitudinal patient monitoring, and molecular tumor boards, with the goal of improving cancer management through enhanced understanding of the entire cancer ecosystem within each patient. We describe this integrative approach using comprehensive analytics generated from serial-biopsied lesions in a metastatic breast cancer patient. The serial biopsies identified remarkable heterogeneity among metastatic lesions that presented clinically as discordance in receptor status and genomic alterations with mixed treatment response. Based on our study, we highlight clinical scenarios, such as rapid progression or mixed response, that indicate consideration for repeat biopsies to evaluate intermetastatic heterogeneity (IMH), with the objective of refining targeted therapy. We present a framework for understanding the clinical significance of heterogeneity in breast cancer between metastatic lesions utilizing multi-omic analyses of serial biopsies and its implication for effective personalized treatment.

SummaryMechanisms of therapeutic resistance manifest in metastatic cancers as tumor cell intrinsic alterations and extrinsic microenvironmental influences that can change during treatment. To support the development of methods for the identification of these mechanisms in individual patients, we present here an Omic and Multidimensional Spatial (OMS) Atlas generated from four serial biopsies of a metastatic breast cancer patient during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata including treatment times and doses, anatomic imaging, and blood-based response measurements to exploratory analytics including comprehensive DNA, RNA, and protein profiles, images of multiplexed immunostaining, and 2- and 3-dimensional scanning electron micrographs. These data reveal aspects of therapy-associated heterogeneity and evolution of the cancer’s genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples showing how integrative analyses of these data provide insights into potential mechanisms of response and resistance, and suggest novel therapeutic vulnerabilities.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

In a pilot study, we evaluated the feasibility of real-time deep analysis of serial tumor samples from triple negative breast cancer patients to identify mechanisms of resistance and treatment opportunities as they emerge under therapeutic stress engendered by poly-ADP-ribose polymerase (PARP) inhibitors (PARPi). In a BRCA-mutant basal breast cancer exceptional long-term survivor, a striking tumor destruction was accompanied by a marked infiltration of immune cells containing CD8 effector cells, consistent with pre-clinical evidence for association between STING mediated immune activation and benefit from PARPi and immunotherapy. Tumor cells in the exceptional responder underwent extensive protein network rewiring in response to PARP inhibition. In contrast, there were minimal changes in the ecosystem of a luminal androgen receptor rapid progressor, likely due to indifference to the effects of PARP inhibition. Together, identification of PARPi-induced emergent changes could be used to select patient specific combination therapies, based on tumor and immune state changes.