RESUMO
Chromobox 2 (CBX2), an epigenetic reader and component of Polycomb Repressor Complex 1 (PRC1), is highly expressed in >75% of high-grade serous carcinoma (HGSC). Increased CBX2 expression is associated with poorer survival, while CBX2 knockdown leads to improved chemotherapy sensitivity. In an HGSC immune competent murine model, knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME), understanding that the TIME plays a critical role in disease progression and development of therapy resistance. Exploration of existing datasets demonstrated that elevated CBX2 expression significantly correlated with the specific immune cell types in the TIME. RNA-seq and pathway analysis of differentially expressed genes demonstrated immune signature enrichment. Confocal microscopy and co-culture experiments found modulation of CBX2 leads to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2 overexpressing cells showed increased M2-like macrophages and decreased phagocytosis activity. Cbx2 knockdown in the Trp53, Brca2 null ID8 syngeneic murine model (ID8 Trp53-/- Brca2-/-) led to decreased tumor progression compared to control. NanoString Immuno-Oncology Panel analysis suggested knock down in Cbx2 shifts immune cell composition, with an increase in macrophages. Multispectral immunohistochemistry further confirmed an increase in macrophage infiltration. Increased CBX2 expression leads to recruitment and polarization of pro-tumor macrophages and targeting CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies.
RESUMO
Despite the success of poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory double-stranded RNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T-cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi-resistant ovarian tumor growth in vivo, and promotes antitumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.
RESUMO
Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.
RESUMO
Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.
RESUMO
High-grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy-resistant disease. PARP inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models, there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-sequencing and an shRNA screen, we identified activating transcription factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence-free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. IMPLICATIONS: This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.
