SCLC cell lines harbour a higher level of both CHK1 gene and protein expression than NSCLC lines. Prexasertib, a CHK1 inhibitor, revealed strong anti-tumour activity in SCLC cell lines, SCLC syngeneic, genetically-engineered mouse (GEM) and chemo-resistant models [48]. The rational of targeting the CHK1/ATR axis in SCLC was confirmed with an independent preclinical study using ATR inhibitors demonstrating activity against SCLC in both in vitro and in vivo models. Promoting ATR through DNA damage leads to many downstream targets like CHK1, which stops cell cycle progression at the G2-M phase. A Phase II trial with Prexasertib in patients with an extended stage (ES)-SCLC was conducted to evaluate its efficacy. It was designed as a parallel-cohort phase II study of 105 mg/m² prexasertib by IV administration. The drug was administered once every 14 days for patients who progressed after no more than two prior lines of therapy and had a platinum-sensitive (Cohort 1) or platinum-resistant/platinum-refractory (Cohort 2) disease. In Cohort 1 (n = 58), ORR was 5.2%; DCR, 31%; median PFS, 1.41 months (95% CI, 1.31–1.64), and median OS, 5.42 months (95% CI, 3.75–8.51). In Cohort 2 (n = 60), ORR was 0%; DCR, 20%; median PFS, 1.36 months (95% CI, 1.25–1.45), and median OS, 3.15 months (95% CI, 2.27–5.52). The most frequent all-grade, related, treatment-emergent adverse events were decreased neutrophil count (Cohort 1, 69.6%; Cohort 2, 73.3%), decreased platelet count (Cohort 1, 51.8%; Cohort 2, 50.0%), decreased white blood cell count (Cohort 1, 28.6%; Cohort 2, 40.0%), and anaemia (Cohort 1, 39.3%; Cohort 2, 28.3%). Eleven patients (19.6%) in Cohort 1 and one patient (1.7%) in Cohort 2 experienced grade ≥3 febrile neutropenia. Prexasertib did not demonstrate enough activity to be considered for future development as monotherapy in ED-SCLC [49].

The anti-tumour activities of PARP inhibitors occur through different mechanisms, including trapping the enzyme to the single-strand DNA breaks (SSBs) by preventing the utilization of nicotinamide adenine dinucleotide (NAD), inhibiting poly ADP-ribosylation (PARylation), as well as binding of PARP to the DNA. Different studies tested PARPs inhibitors either as a single agent or in combination with other treatments. As their single-agent activity is limited, a series of clinical studies examined various combinations of PARP inhibitors with chemotherapy, radiation, and targeted therapies to increase their therapeutic benefit in this hard-to-treat disease. Owonikoko et al. tested the combination of veliparib with cisplatin (75 mg/m²) and etoposide (100 mg/m² on days 1–3) in phase I/II randomized clinical trial (ECOG-ACRIN 2511), including patients with ES-SCLC. Patients with ES-SCLC, stratified by sex and serum lactate dehydrogenase levels, were randomly assigned to receive four three-week cycles of cisplatin-etoposide (CE) (75 mg/m² intravenously on day 1 and 100 mg/m² on days 1 through 3) along with veliparib (100 mg orally twice per day on days 1 through 7) or placebo (CE+P). The primary endpoint was PFS. The respective median PFS for the CE+V arm vs. the CE+P arm was 6.1 vs. 5.5 months (unstratified HR 0.75 [one-sided p = 0.06]; stratified HR, 0.63 [one-sided p = 0.01]), favouring CE+V. The mOS was 10.3 vs. 8.9 months (stratified HR, 0.83; 80% CI, 0.64 to 1.07; one-sided p = 0.17) for the CE+V and CE+P arms, respectively. The ORR was 71.9% vs. 65.6% (two-sided p = 0.57) for CE+V and CE+P, respectively. The following grade ≥ 3 haematology toxicities were more frequent in the CE+V arm than the CE+P arm: CD4 lymphopenia (8% vs. 0%; p = 0.06) and neutropenia (49% vs. 32%; p = 0.08), but treatment delivery was comparable. The addition of veliparib to frontline chemotherapy showed a signal of efficacy in patients with ES-SCLC, and the study met its prespecified end point [50].

Two phase II studies in relapsed SCLC patients evaluated the combination of temozolomide (TMZ) and PARP inhibition. Pietanza et al. performed a randomized, double-blind, placebo-controlled study of either veliparib (40 mg twice daily, days 1 to 7) or placebo and TMZ (150–200 mg/m²/day, days 1 to 5) on a 28-day cycle [51]. As a primary endpoint, the study had four-month PFS, with no significant differences observed between TMZ/veliparib (36%) and TMZ/placebo (27%, p = 0.19). Median PFS was 3.8 and 2.0 months (log-rank p = 0.39, HR 0.84; 95% CI: 0.56 to 1.25) for the TMZ/veliparib and TMZ/placebo arms, respectively. OS was also similar between the two arms. Instead, ORR has been shown to be higher for the combination of TMZ/veliparib (39%) vs. TMZ/placebo (14%) in both platinum-sensitive and platinum-refractory patients.

Using another PARP inhibitor, olaparib, in combination with TMZ, Farago et al. performed a phase I/II study in relapsed SCLC [52]. At the recommended phase II dose of olaparib (200 mg twice daily, day 1–7) and TMZ (75 mg/m², day 1–7 of 21 days cycle), the ORR was 41%, with a median duration of response of 5.3 months. Across all dose levels, PFS was 4.2 months (95% CI, 2.8 to 5.7) with a median OS of 8.5 months (95% CI, 5.1 to 11.3). Another phase II study with continuous talazoparib associated with intermittent low-dose TMZ (NCT03672773) in relapsed/refractory SCLC is currently ongoing.

PARP inhibitors activity was also studied in combination with immunotherapy, based on a potential synergistic activity between such different approaches. A phase II trial in relapsed SCLC combining durvalumab 1500 mg every four weeks with olaparib 300 mg twice a day demonstrated an ORR of 10.5% (two patients out of nineteen) [53]. Similar results have been recently reported from the phase I/II multicentre open-label and single-arm basket MEDIOLA trial. From May 2016 to December 2016, 40 patients with limited or extended relapsing SCLC were enrolled. They received olaparib monotherapy (300 mg twice daily) for four weeks, followed by a combined treatment of olaparib (300 mg twice daily) and durvalumab 1500 mg iv administered every four weeks. ORR resulted in 10.5% (95% CI: 2.9–24.8). Meanwhile, mPFS was 2.4 months (95% CI: 0.9–3.0), and OS resulted in 7.6 months (95% CI: 5.6–8.8). Even if the study failed to reach the primary endpoint of disease control rate at 12 weeks (28.9%), one patient achieved a complete response and three a partial response. Grade 3 or higher adverse events were reported in 32 patients (80%), with anaemia (40%) and lymphopenia (12.5%) being the most frequent. These data suggested a limited activity of PARP inhibitors in SCLC patients, while additional studies are investigating the potential role of this therapeutic strategy in selected populations [54].

Another interesting approach might be the combination of ATM/ATR inhibitors together with the topoisomerase I inhibitor topotecan. A phase 2 trial was designed with a combination of Berzosertib (M6620), an ATP-competitive ATR inhibitor, and topotecan in SCLC patients who had relapsed after at least one prior chemotherapy. The primary endpoint was ORR. M6620 (210 mg/m² intravenously on days 2 and 5) was administered concurrently with topotecan (1.25 mg/m² intravenously on days 1 through 5) in 21-day cycles. A total of 26 patients were enrolled, and all of them had evidence of disease progression before study participation. Seven of 16 (43.8%) patients showed a partial response (PRs) in the first stage, permitting the continuation of enrolment in the second stage. In the overall study, 9 of 25 patients (36.0%, 95% CI: 18.0–57.5) obtained a confirmed partial response, reaching the primary endpoint for a response. Most patients (17/25 patients; 68.0%) obtained tumour regressions. After a median potential follow-up of 20.7 months, the median PFS was 4.8 months (95% CI: 2.8–7.4). The PFS at 4 and 6 months was 60.0% (38.4–76.1) and 36.0% (18.2–54.2), respectively. The median OS was 8.5 months (5.6–13.6), and OS at 6 and 12 months was 68.0% (46.1–82.5) and 32.0% (15.2–50.2), respectively. Responses were achieved in patients with both platinum-sensitive and platinum-resistant disease. These trial results provide evidence to support the strategy of a mixed ATR and TOP1 inhibition in order to empower the topotecan efficacy in SCLC patients [55].

Inhibition of Aurora kinase A or B arrests the proliferation and growth of both in vitro and in vivo SCLC models [56]. In a recently reported clinical trial, an aurora kinase A inhibitor, alisertib, combined with paclitaxel, had significantly improved PFS compared to paclitaxel alone in patients with cMYC-positive SCLC. The efficacy of targeting AURKA was studied in a randomized phase II study of paclitaxel plus alisertib vs. paclitaxel plus placebo as second-line therapy. In this double-blind study, patients affected by relapsed or refractory SCLC were stratified considering the relapse pattern (sensitive vs. resistant or refractory) as well as the presence of brain metastases and randomized 1:1 to alisertib/paclitaxel or placebo plus paclitaxel. A total of 178 patients were enrolled (89 in each arm). The median PFS was 3.32 months with alisertib/paclitaxel vs. 2.17 months with placebo/paclitaxel (HR = 0.77), thus confirming a promising activity of alisertib/paclitaxel in relapsed or refractory SCLC [57].

In SCLC, there are common inactivating mutations in the primary Notch family members and overexpression of a key negative regulator of Notch signalling known as delta-like protein 3 (DLL3) was found in the majority of SCLC tumours [58]. Initial clinical evaluation of an anti-DLL3 antibody-drug conjugate rovalpituzumab tesirine (Rova-T) had promising activity, although this agent was comprised of several toxicities [59]. Rovalpituzumab tesirine (Rova-T) has the structure of an antibody-drug conjugate containing a DLL3-targeting antibody joined to a cytotoxic agent, pyrrolobenzodiazepine. The efficacy and safety of Rova-T compared with topotecan as second-line therapy were evaluated in patients with SCLC expressing high levels of DLL3 (DLL3-high). The TAHOE study was an open-label, two-to-one randomized, phase 3 study comparing Rova-T with topotecan. The setting of this study was the second-line therapy in DLL3-high advanced or metastatic SCLC. Rova-T (0.3 mg/kg) was given intravenously on day 1 of a 42-day cycle for two cycles, with two additional cycles available for specific patients. Topotecan (1.5 mg/m²) was administered intravenously on days 1 to 5 of a 21-day cycle. Patients randomized to Rova-T (n = 296) and topotecan (n = 148) were included in the efficacy analyses. The median OS was 6.3 months (95% CI: 5.6–7.3) in the Rova-T arm and 8.6 months (95% CI: 7.7–10.1) in the topotecan arm (HR, 1.46 [95% CI: 1.17–1.82]). An independent data monitoring committee stated that enrolment had to be discontinued because of the shorter OS observed with Rova-T compared with topotecan. Safety profiles for both drugs were not different from previous reports. Compared with topotecan, the current standard of care for second-line chemotherapy, Rova-T demonstrated an inferior OS and higher rates of side effects. These effects were represented by serous effusions, photosensitivity reactions, and peripheral oedema. Despite this failure, other trials are currently evaluating anti-DLL3 efficacy in SCLC.

Tarlatamab, a bispecific T-cell engager molecule (BiTE), in patients with relapsed/refractory SCLC, was evaluated in a phase 1 study. The primary end point was safety. Secondary end points included antitumor activity by modified RECIST 1.1, overall survival, and pharmacokinetics. By 19 July 2022, 107 patients received tarlatamab within both dose exploration (0.003 to 100 mg; n = 73) and expansion (100 mg; n = 34) cohorts. Median prior lines of anti-cancer therapy achieved by patients were 2 (range, 1–6); 49.5% received anti-programmed death-1/programmed death ligand-1 therapy. Any-grade treatment-related adverse events occurred in 97 patients (90.7%) and grade ≥ 3 in 33 patients (30.8%). One patient (1%) experienced grade 5 pneumonitis. Cytokine release syndrome was the most common treatment-related adverse event, occurring in 56 patients (52%), including grade 3 in one patient (1%). The maximum tolerated dose was not reached. The objective response rate was 23.4% (95% CI, 15.7 to 32.5), including two complete and 23 partial responses. The median duration of response was 12.3 months (95% CI, 6.6 to 14.9). The disease control rate was 51.4% (95% CI, 41.5 to 61.2). The median PFS and OS were 3.7 months (95% CI, 2.1 to 5.4) and 13.2 months (95% CI, 10.5 to not reached), respectively. Exploratory analysis suggests that selecting for increased DLL3 expression can result in increased clinical benefit [60]. In patients with heavily pre-treated SCLC, tarlatamab showed manageable safety with promising response durability. Further evaluation of this promising molecule is ongoing in the context of prospective randomized clinical studies.

Lurbinectedin, a DNA binding agent that seems to work as a selective inhibitor of RNA polymerase II transcription, demonstrated substantial activity against SCLC [61]. This drug induces selective degradation of RNA pol. II leading to apoptosis in tumour cells. The evidence of lurbinectedin activity in SCLC derives from a cohort of a single-arm, open-label, phase II basket trial conducted by Trigo et al. [62]. The authors enrolled 105 patients with advanced SCLC pre-treated with only one previous line of treatment (IO was allowed alone or in combination with CHT) and Eastern Cooperative Oncology Group (ECOG) performance status of two or lower. According to the investigator’s assessment, after a median follow-up of 17.1 months, the study reached its primary endpoint with a RR of 35.2% (95% CI: 26.2–45.2) in the entire cohort. In a pre-planned conducted analysis, the overall responses were higher in patients with sensitive disease compared with resistant disease. Of note, 60.9% and 27.1% of patients were still alive after one and two years, respectively. When considered together, these data are very important in terms of response and survival, if compared with historical controls, in both groups of patients with resistant and sensitive diseases. Furthermore, lurbinectedin had a good safety profile with manageable toxicity. After the positive results of this phase II study, on June 2020, lurbinectedin received approval from the FDA for patients with SCLC in progression on or after platinum-based CT and has recently been granted orphan drug status by the European Medicine Agency (EMA). Lurbinectidin was later evaluated in a phase III trial [63]. Atlantis study is an open-label, randomized, multicenter phase III trial testing the second-line efficacy of the combination of lurbinectedin and doxorubicin compared to the investigator’s choice of CT with CAV (cyclophosphamide/doxorubicin/vincristine) or topotecan. In this study were enrolled pre-treated patients with histologically confirmed diagnoses of limited or ED SCLC whose disease progressed after one prior platinum-containing line. Its first endpoint, the OS, was not reached.

In conclusion, lurbinectedin has demonstrated good activity as a single agent in second-line therapy of SCLC, to a large extent in platinum-sensitive patients, but failed to exhibit an improvement in OS when combined with doxorubicin compared with CAV or topotecan. Although the primary endpoint of OS in the phase III study was not reached, lurbinectedin plus doxorubicin showed a good safety profile. Lurbinectedin is a treatment option for patients progressing on or after first-line platinum-based ChT [13,64].

Several randomized trials tested the anti-VEGF monoclonal antibody, bevacizumab, in combination with standard chemotherapy in SCLC patients, showing poor results and no clear survival benefits [65,66]. A randomized phase III, open-label, multicentre clinical trial enrolled 205 patients with ED-SCLC, investigating bevacizumab in combination with etoposide and cisplatin in the first line. At a median follow-up of 34.9 months, an improvement of median OS (9.8 vs. 8.9 months; HR = 0.78), 1-year survival rates (37% vs. 25%) and objective response (58.4% vs. 55.3%) has been observed in favour of bevacizumab-treated patients.

Sorafenib combined with chemotherapy was reported to have significant toxicity and low efficacy in a phase 2 trial [67]. A total of 18 patients were enrolled, with 17 evaluable patients. One patient had a complete response, seven patients had a partial response (overall response rate of 47%), and one patient had stable disease. Median OS was 7.4 months, and one-year survival was 25%. The most common treatment-related adverse events included fatigue, anorexia, rash, diarrhoea, neutropenia and weight loss. Grade 5 gastrointestinal bleeding, pulmonary haemorrhage and neutropenia occurred in one patient (6%) each. Accrual was halted on the basis of the safety profile as well as preliminary efficacy data. The combination of platinum-based chemotherapy and sorafenib has significant toxicity at current dose levels and is associated with disappointing efficacy data.

Thalidomide is another anti-angiogenic drug that was evaluated in SCLC [68]. In a phase 3 trial, thalidomide combined with chemotherapy did not improve survival in SCLC patients with limited disease or extensive disease [69]. Thalidomide was also investigated both in combination with carboplatin-etoposide and as maintenance therapy in patients with untreated SCLC. Median progression-free and overall survival were 8.3 months and 10.1 months, respectively. One-year survival was 40%, and the one-year progression-free survival was 36%. The ORR was 68% (95% CI 46–85%), with four complete remissions (20%) and 13 partial remissions (48%). No increase in chemotherapy-related toxicity was observed. Thalidomide was well-tolerated, and the median time on thalidomide treatment was 7.6 months.

Differently from other VEGF inhibitors, apatinib, a selective target of VEGFR2, demonstrated good results in previous studies and also in SCLC settings. A phase II trial showed acceptable toxicity in pre-treated patients receiving apatinib. Forty patients were enrolled. At the data cut-off time (15 November 2018), the median follow-up was 7.4 months; no patients remained on treatment, and five were still in follow-up. An objective response was achieved in 7 of 40 patients (17.5%) in the intention-to-treat population and 7 of 38 patients (18.4%) in the per-protocol population. The median PFS and OS were 3.0 months and 5·8 months, respectively. The most commonly observed grade 3 or greater treatment-related adverse events were hypertension, hand-foot syndrome, and increased L-gamma-glutamyltransferase [70]. Apatinib exhibited efficacy and an acceptable safety profile in previously heavily-treated ES-SCLC patients. Further exploration of apatinib in phase III trials is warranted.

Similarly to apatinib, another angiogenic multikinase inhibitor, anlotinib, exhibiting activity against VEGFR 1-2-3, FRGR 1-4, PDGFR a/b and c-Kit, has shown encouraging results. From 2017 to 2018, a prospective randomised, double-blind trial was conducted to evaluate its efficacy (versus placebo) in patients affected by SCLC failing at least two prior lines of treatment. The study demonstrated a PFS advantage over placebo: 4.1 months (95% CI: 2.8–4.2) vs. 0.7 months (95% CI: 0.7–0.8), with an acceptable toxicity profile. To date, several clinical trials are currently ongoing to evaluate its efficacy and safety in different settings [71].

Since the human epigenome could be visualized using next-generation sequencing, the role of epigenetic processes in SCLC could be understood [63]. The most promising epigenetic regulatory proteins are enhancers of zeste homolog 2 (EZH2) and lysine-specific demethylase 1A (LSD1). Both of them are now being tested in SCLC clinical trials. Respectively two promising drugs against EZH2 and LSD1 tested in SCLC are Tazemetostat and GSK2879552. EZH2 itself is a common target of deregulated expression in cancers. Aberrant EZH2 expression in cancers is due to genetic, transcriptional, post-transcriptional, and post-translational modifications [72]. EZH2 inhibitors are mostly tested with platinum-based compounds but are also being explored in the context of combination regimens, including docetaxel, etoposide, temozolomide (chemotherapy), PD-L1 and PD-1 inhibitors, antiandrogens, PARP and HDAC inhibitors. These are expected to increase the effects of EZH2-targeted therapy. Gardner et al. showed that chemoresistance to cisplatin and etoposide in SCLC is partially due to the suppression of SLFN11, a protein with the role of inhibiting DNA replication and promoting cell death after DNA damage. The authors showed that EZH2 interacting with SLFN11 promotes chemoresistance [73]. Elements that definitely demonstrate the concrete activity of anti-EZH2 in SCLC are still immature and under investigation.