Combination of PARP and DNA methylation inhibitors as a potential personalized therapy for SETD2-mutated clear-cell renal cancers

Clear-cell renal cell carcinoma (ccRCC) arises from proximal tubules in the kidney cortex and is the most common kidney malignancy (1). Metastasis occurs in over one-third of cases and targeted therapies, primarily against angiogenesis, have been used in clinical practice over the past decade (1). The application of anti-angiogenesis therapies has been motivated by the inactivation of Von Hippel-Lindau (VHL) tumor suppressor gene, which is the most common genetic alteration in ccRCC. VHL deficiency leads to the aberrant activation of hypoxia signaling and downstream angiogenesis pathways (2,3). Furthermore, due to the immunogenic nature of ccRCC tumors, immune checkpoint inhibitors (ICIs) are used as the standard of care for advanced tumors, most recently in combination with tyrosine kinase inhibitors (TKIs) (3,4). While some patients benefit from these treatments, responses to anti-angiogenesis and ICI agents are transient and limited to a small subset of patients, emphasizing the critical need for prognostic and predictive biomarkers, which are currently lacking in clinical practice (5-7). Notably, the current therapies are directed against tumor microenvironment, highlighting the challenge of targeting cancer cells directly within ccRCC tumors.

Recent large-scale sequencing studies have advanced our knowledge about genomic drivers of ccRCC beyond VHL mutations and revealed significant heterogeneity in the genomic landscapes of tumors, which is linked to diverse clinical outcomes (8,9). The most commonly mutated genes in ccRCC include VHL (76%), PBRM1 (39%), SETD2 (18%), and BAP1 (14%), all located on the short arm of chromosome 3 (8,10). Interestingly, PBRM1 and SETD2 encode two prominent epigenome modulators, highlighting the driving role of abnormal epigenome patterns in ccRCC development. In this context, previous studies have reported the anti-proliferative activity of DNA methyltransferase inhibitor 5-aza-20-deoxycytidine (DAC) against ccRCC cells (11). Notably, the combination of DAC with chemotherapeutic agents effectively demonstrates a synergistic impact in reducing the viability of renal cell carcinoma (RCC) cell lines (12). SETD2 is the leading histone methyltransferase, responsible for catalyzing the methylation of histone-3 at lysine-36 (H3K36me3), and according to a previous study, SETD2 deficiency can increase DNA double-strand breaks (DSB) damage by impairing homologous recombination (HR) (13). These observations have inspired Zhou and colleagues to examine the potential impacts of combination therapy with DNA hypomethylating agents (HMAs) and DNA repair inhibitors in ccRCC. Specifically, they evaluated the effects of combining DAC with an inhibitor of poly (ADP-ribose) polymerase (PARP) (talazoparib; BMN-673) on the viability and growth of SETD2-deficient human ccRCC (14).

PARP inhibitors (PARPi) interrupt the ability of malignant cells to tolerate DNA damage, leading to cell cycle arrest and apoptosis, and have shown significant efficacy in different tumor types (15).

Zhou and colleagues investigated the effects of treatment with BMN-673 and DAC on different RCC cell lines, categorized into three groups as follows: (I) ACHN (SETD2 wild-type) and A498 (SETD2 mutant); (II) Caki-2 (SETD2 wild-type) and 769-P (SETD2 down-regulated); (III) 786-O SETD2 wild-type (SETD2-WT) and 786-O SETD2 knock-out (SETD2-KO). First, the authors observed a synergetic inhibition of cell growth by inducing apoptosis in SETD2-mutated cells upon a combination therapy with DAC and BMN-673. Moreover, they showed a relationship between increased cell cycle inhibition and apoptosis induction and higher rates of DNA damage, inefficient DNA repair systems for DNA DSBs, and loss of genomic stability in SETD2-altered cells.

Second, they employed RNA-sequencing to examine alterations in transcriptional profiles of 786-O SETD2-WT and 786-O SETD2-KO cells before and after treatments. The Gene Set Enrichment Analysis (GSEA) revealed that main pathways upregulated following treatments with DAC alone or in combination with BMN-673 were related to innate and adaptive immune responses. The activation of immune response pathways correlated with an increased expression of transposable elements (TEs), particularly following the combination therapy. However, these pathways were not significantly affected following PARPi monotherapy, indicating a noticeable effect of the dual treatment on the activation of immune-related pathways. Additionally, the study revealed a significant increase in STING1 protein levels, particularly in SETD2-KO cells, following treatment with DAC or the combination of DAC and BMN-673. These findings suggest that activation of TEs, resembling viral mimicry, and the STING1 pathway are potentially involved in immune response activation and may contribute to the therapeutic impacts observed after treatment with DAC or the combination of DAC and BMN-673.

Lastly, to validate in vitro findings, Zhou et al. examined the effects of therapies in vivo on xenografts developed by implanting SETD2-WT and SETD2-KO 786-O cells in immune-deficient nude mice. The in vivo results confirmed that the SETD2-deficient xenografts were more sensitive to the combination therapy, consistent with the in vitro results (14).

The results of this study are novel and interesting in terms of proposing a new therapeutic strategy that targets cancer cells directly in ccRCC tumor milieu. However, the translational potential of the findings cannot be assessed thoroughly due to the limitations of the study, as discussed below. First, the results are generated using a limited number of cell lines in vitro and only one cell line (786-O) in vivo. Given the diverse histological subtypes of RCC, the in vivo observations are only limited to the ccRCC subtype (represented by 786-O) and may not be valid for other SETD2-mutant subtypes of RCC. Second, the cell line models do not capture the complex environment of tumors faithfully, and patient-derived organoid (PDO) or patient-derived xenograft (PDX) models have emerged as more appropriate tools for the evaluation and pre-clinical studies of novel treatment approaches (16). Lastly, the use of immune-deficient animals did not allow to verify possible involvement of immune responses.

Genome-based therapeutic approaches in RCC have a long-standing history. An eminent example is the application of anti-angiogenesis TKIs in ccRCCs, characterized by inactivating mutations of VHL. While the use of anti-angiogenic agents was the first-line treatment in ccRCC for many years, the landscape of ccRCC management is consistently changing, owing to the emergence of new links between genomic alterations and targeted therapies. For example, it has been proposed that PBRM1 mutations may be associated with better response to ICI treatments (17,18). Furthermore, recent research has shown that RCC cells carrying SETD2 mutations or exhibiting reduced expression of SETD2 are sensitive to HMAs both in vitro and in vivo (19). In addition, it has been shown that combination therapy with an HMA and PARPi has the potential to inhibit cell growth in some cancers (20,21). In this context, the study by Zhou et al. is the first report proposing a dual therapy with DAC and PARPi against SETD2-mutant ccRCC (14). The results of this study can complement those of preclinical studies that have indicated the potential use of PARPi in ccRCC management (22,23), and a current phase Ib/II clinical trial that is evaluating the outcomes of the talazoparib and axitinib combination in patients with previously treated metastatic RCC (24). In sum, the study by Zhou et al. may open new avenues for personalizing PARPi-therapies for SETD2-mutated metastatic ccRCCs, addressing the limitations of existing approaches (25). However, additional studies are needed to validate these observations and elucidate the involved pathways.

Acknowledgments

Funding: None.

Footnote

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