Targeting the EZH2-SLFN11 pathway—a lesson in developing molecularly-informed treatments for recurrent small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive, high-grade neuroendocrine pulmonary tumor comprising 14% of lung cancer diagnoses, with approximately two-thirds of patients presenting with extensive-stage disease, generally considered to be incurable (1). While the incorporation of checkpoint inhibitor immunotherapy alongside platinum and etoposide chemotherapy represents a major therapeutic advance for extensive-stage disease, relative survival improvement over chemotherapy is modest, and median overall survival (OS) remains around 12 to 13 months (2,3). Thus, more effective therapeutic options in the relapsed setting remains a critical area of unmet need.

Both U.S. and European clinical practice guidelines recommend considering the chemotherapy treatment-free interval to determine platinum-sensitive versus platinum-refractory status in considering candidacy for retreatment with platinum doublet chemotherapy, reflecting the historical dearth of subsequent options (4,5). Otherwise, if a clinical trial is not available or feasible, U.S. Food and Drug administration (FDA) approved treatment options include topotecan (a topoisomerase I inhibitor), lurbinectedin (an alkylating agent and inhibitor of oncogenic transcription), and most recently tarlatamab (a bispecific T-cell engager targeted at DLL3 and CD3) (6). Irinotecan, while not FDA-approved, is routinely used as an alternative to topotecan in clinical practice.

While the characteristic inactivation of tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) has been known for many years, the identification of predictive biomarkers in SCLC lags behind its non-small cell lung cancer (NSCLC) counterpart (7). Among recent developments was the observation that Schlafen family member 11 (SLFN11) is expressed in approximately half of extensive-stage SCLC (ES-SCLC), though thresholds for positivity vary (8,9). SLFN11 is thought to encode a DNA/RNA helicase involved in the DNA damage response. Its expression has been correlated with sensitivity to several cytotoxic chemotherapies and poly (ADP-ribose) polymerase (PARP) inhibitors in vitro (10). Exploratory biomarker analysis in a phase 2 trial of the oral alkylating agent temozolomide combined with the PARP inhibitor veliparib demonstrated improved survival for SLFN11-positive compared to SLFN11-negative SCLC, which was not true of the temozolomide plus placebo arm (8). Furthermore, downregulation of SLFN11 through epigenetic silencing, specifically Enhancer of Zeste homolog 2 (EZH2), has been identified through SCLC patient derived xenograft (PDX) models as a potential method of acquired chemoresistance to carboplatin plus etoposide in a single study (11). Notably, while treatment with EZH2 inhibition in PDX models restored SFLN11 expression and chemosensitivity in SLFN11^HIGH models, SLFN11^LOW tumors did not exhibit either of these effects, suggesting that the strategy of EZH2 inhibition to rescue chemoresistance may apply to only a subset of SCLC (11).

Thus forms the clinical background and preclinical basis for the trial conducted by Choudhury et al. (12). This was a single-center, open-label, single-arm phase I/II trial assessing the EZH1/2 inhibitor valemetostat with fixed-dose irinotecan 125 mg/m² on days 1 and 8 of a 21-day cycle. Valemetostat was initiated 7 days prior to irinotecan, functioning as a run-in phase to assess the tolerability of single-agent valemetostat. The phase I portion of the trial followed a typical 3+3 design. The primary endpoint of the phase II portion was objective response rate (ORR) at the recommended phase 2 dose (RP2D). Patients were required to have disease progression after prior platinum-etoposide chemotherapy (with or without immunotherapy), but could have either platinum-refractory (defined as start of second-line treatment less than 90 days after end of first-line therapy) or platinum-sensitive disease. Patients with untreated or symptomatic central nervous system (CNS) metastases were excluded.

As with any single-arm uncontrolled study, the underlying assumptions and statistical considerations must be carefully evaluated in the context of existing data. The study was powered to detect a 9% improvement in ORR for the combination of valemetostat plus irinotecan, assuming a single-agent irinotecan response rate of 16%. Single-agent irinotecan has been evaluated in several small phase 2 studies at varying doses ranging from 100 mg/m² on days 1 and 8 every 3 weeks to 300 mg/m² once every 3 weeks. Response rates in these trials of relapsed SCLC range from 0 to 47%, with variability likely explained by heterogeneity of the recruited populations, particularly platinum-sensitivity (13-16). While the phase 2 study referenced by Choudhury et al. appears to be published in abstract form only, the assumed single-agent ORR for irinotecan remains reasonable (17).

The trial was terminated after enrollment of 22 patients due to pre-specified early stopping rules for toxicity, with myelosuppression and associated infectious complications among the most common grade 3 and 4 treatment-related adverse events (AEs). Fatigue and gastrointestinal AEs (diarrhea, nausea, abdominal pain) were frequent, including multiple grade 3 events. Dose reduction of both irinotecan and valemetostat were common, and 3 patients (14%) discontinued treatment for toxicity, including 1 patient who never started irinotecan due to thrombocytopenia during the valemetostat run-in period. Ultimately, the overlapping toxicities of combination valemetostat and irinotecan led to poor tolerance. Lower-dose irinotecan (60 mg/m² on days 1, 8, and 15 every 4 weeks) has been studied in an effort to mitigate the frequent gastrointestinal toxicities and myelosuppression of standard-dose irinotecan with improvement in toxicity profile, but is not standardly used (18). Valemetostat is approved in Japan for relapsed/refractory adult T-cell leukemia/lymphoma based on a phase 2 trial where toxicities were mainly hematologic, including grade 3 or higher treatment-emergent anemia and thrombocytopenia in one-third of patients (19). While this comparator is in a different malignancy and at a higher dose (200 mg daily, compared to 100 mg daily as the RP2D in the trial by Choudhury et al.), it is not surprising that the dose-limiting toxicities of combination valemetostat and irinotecan were mainly hematologic. Ongoing development of third-generation PARP inhibitors with increased potency and attenuated hematologic toxicity may make such combinations more tolerable and clinically feasible.

In aggregate, it is challenging to conclude much from the observed ORR of 21%, median progression-free survival (PFS) of 2.2 months, and median OS of 6.6 months among the 19 patients evaluable for response. Due to early termination, the study was underpowered for its efficacy endpoint, with findings overall similar to the outcomes for single-agent irinotecan across multiple studies (13,14,16). Notably, the authors did highlight three patients who were able to remain on treatment for more than 7 months, two of whom seemed to convert dominant subtypes, with the caveat that biopsies were from different sites of metastatic disease. Further analysis and characterization of these responders would be interesting to pursue to determine patients most likely to benefit from this strategy.

More interesting are the correlative biomarker studies conducted by these authors, asking the question of whether EZH2 inhibition via valemetostat can restore SLFN11 expression and chemosensitivity. An important limitation in interpreting the subsequent findings is that repeat tissue biopsy was not required at enrollment, meaning archival tumor biopsies were allowed as pre-treatment specimens. It is not clear from the presented data how many of the tissue samples were obtained from the treatment-naïve setting versus after progression on platinum-etoposide chemotherapy. Given the preclinical hypothesis that SLFN11 depletion is involved in chemoresistance of SCLC, the distinction between the treatment-naïve and post-chemotherapy time points is critical. It is difficult to make any conclusions about the effect of EZH2 inhibition on SLFN11 or MHC-I expression in this study, as we lack a clear understanding of the baseline state immediately prior to this line of therapy. Furthermore, as SLFN11 expression quantified by the H-score is a continuous variable, we also need a better understanding of what are clinically meaningful cut-off points and absolute differences.

The current transcription-factor based molecular classification of SCLC subdivides the disease into four subtypes: SCLC-A (ASCL1), SCLC-N (NEUROD1), SCLC-P (POU2F3), and SCLC-I (characterized as an inflamed subtype lacking ASCL1, NEUROD1, and POU2F3 expression) (20,21). These categories have been proposed as a framework to predict specific therapeutic vulnerabilities. While this remains an area of active investigation and validation, an example of this in the clinical setting involved retrospective analysis of RNA sequencing data from the phase 3 IMpower133 trial, showing that patients with the SCLC-I subtype had a trend towards improved OS with first-line chemotherapy plus atezolizumab, which was not true of the chemotherapy plus placebo arm (21). Other proposed subtype-specific vulnerabilities based on in vitro drug response data include: (I) PARP inhibitor and platinum sensitivity in the SCLC-A subtype if SLFN11 positive, (II) PARP inhibitor sensitivity in the SCLC-P subtype, independent of SLFN11 expression, and (III) ATR pathway inhibition as a mechanism to overcome chemoresistance in SLFN11 negative cell lines (21,22). Based on these putative vulnerabilities, the multicohort phase 2 S2409-PRISM study is under development through the Southwest Oncology Group, with the goal of testing checkpoint inhibitor immunotherapy versus several biomarker-directed targeted agents with immunotherapy in the maintenance setting based on SCLC subtypes and SLFN11 expression (23). Notably, a recent phase 2 trial was conducted in patients with newly-diagnosed SLFN11-positive ES-SCLC after frontline chemoimmunotherapy, randomizing patients to maintenance atezolizumab plus talazoparib versus standard atezolizumab maintenance. Results indicated a statistically significant PFS benefit for the combination group [hazard ratio (HR) 0.66, P=0.019], though with questionable clinical significance (median PFS 2.9 versus 2.4 months) and no OS difference (24).

Intratumoral heterogeneity for SCLC subtypes has been demonstrated in patient-derived tumor samples, although the prognostic and predictive significance of these findings at a single time point are not yet clear (21). Furthermore, the temporal evolution of transcription factor defined subtypes with systemic treatment, though increasingly reported, remain primarily hypothesis-generating (25). While some studies have suggested general conservation of subtypes with chemotherapy, single-cell analyses suggest small populations of tumor cells demonstrate subtype evolution with treatment (21,26). This then raises the technical distinction between assessing transcription factor expression in aggregate via immunohistochemistry versus at the RNA transcript and single-cell level via advanced sequencing techniques. With this in mind, the study by Choudhury et al. provides the paired pre- and on-treatment subtypes for 7 patients, recognizing that location of biopsy site varied for 3 of these patients. Of note, the authors include YAP1 expression as a method of subtyping, the validity of which has been debated since its initial report (20,21). While an interesting observation, the finding that the patients with the longest duration of response demonstrated subtype switching requires further validation, both due to the small sample size and unclear timing of baseline biopsy.

In summary, the trial by Choudhury et al. is a commendable effort to translate preclinical hypotheses about chemoresistance in recurrent SCLC into a molecularly-informed clinical protocol. As this study was terminated early for toxicity, it remains unknown whether EZH2 inhibition and more broadly, epigenetic regulation, has a role in the treatment of SCLC. Aside from alternative combinations or dosing regimens, whether EZH2 inhibition could have a role in the maintenance setting warrants further investigation. Patient selection might be optimized by screening for tumors with SLFN11 expression at baseline, given that in preclinical models EZH2 inhibition restored chemosensitivity in only SLFN11^HIGH tumors (11).

The main limitation in terms of translational relevance is the unclear timing of pre-treatment tissue biopsy relative to the therapies a patient received. We recognize that requiring fresh tissue biopsy at the time of clinical trial enrollment for recurrent SCLC is cumbersome and may not be feasible depending on the tempo of progressive disease. For this reason, developing technologies such as the use of DNA methylation patterns from circulating tumor DNA (ctDNA) in liquid biopsies to predict SCLC subtypes may allow more convenient longitudinal tracking of tumor evolution with treatment (27).

Molecular subtyping as a method of understanding and overcoming chemoresistance in SCLC is an important area of active investigation. The design of prospective clinical trials based on potential predictive biomarkers is critical, though the correlative specimens collected should ideally be able to validate the proposed biologic mechanism. Future research efforts directed at standardizing definitions for existing biomarkers and optimizing technologies such as subtyping by liquid biopsy will be imperative.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Translational Cancer Research. The article has undergone external peer review.

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1755/prf

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1755/coif). F.S. has participated in an advisory board for OncoHost and received honoraria from MJH Life Sciences. M.S.D. has received honoraria from Plexus Communications, IDEO Health, Springer, Medical Educator Consortium, Dedham Group, DAVA Oncology, MJH Life Sciences, Targeted Oncology, OncLive, Association of Northern California Oncologists, Aptitude Health, MashUp Media, Curio Science, Med Learning Group, Triptych Health, and American Cancer Society; has participated in an advisory board for Advarra; has served as a consultant for Sanofi/Genzyme, Regeneron, Catalyst Pharmaceuticals, Novocure, Guardant, EMD Soreno, Janssen, Abbvie, Gilead, Daiichi Sankyo, and Bristol Myers Squibb; and has received institutional research funding from Merck, Genentech, CellSight, Novartis, and Varian. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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