NOTCH1 Boosts SCLC Survival Rates by 30%

High NOTCH1 expression is significantly associated with longer overall survival with the addition of an anti–PD-L1 inhibitor to first-line chemotherapy among NE subsets of extensive-stage SCLC patients. Given our previous work demonstrating an association between Notch signaling and clinical benefit with ICB in relapsed SCLC (20), we hypothesized that there may be a relationship between Notch signaling and overall survival (OS) among patients with first-line ICB-treated extensive-stage SCLC. To test this hypothesis, we performed an unbiased generalized random forest analysis using the 32 genes of the Hallmark Notch signaling gene set (https://www.gsea-msigdb.org/gsea/msigdb) within the NE-enriched subset (NMF1/2/3) of IMpower133 previously shown to have longer OS with the addition of atezolizumab (anti–PD-L1 inhibitor) to chemotherapy than with placebo plus chemotherapy (18). Among the Hallmark Notch signaling genes, the model identified NOTCH1 as the top gene that may be predictive of OS with atezolizumab over placebo (Figure 1A). Further analysis demonstrated that in this NE-enriched subset, high (defined as greater than or equal to median) NOTCH1 expression was associated with significantly longer OS with atezolizumab compared with placebo (HR 0.53; 95% CI, 0.34–0.81; unadjusted P = 0.003) (Figure 1B), whereas low NOTCH1 expression was not (HR 0.80; 95% CI, 0.51–1.24; unadjusted P = 0.31) (Figure 1B). In contrast, in the non-NE-enriched subset previously shown to lack an OS benefit with the addition of atezolizumab to chemotherapy (18), there were no significant differences in OS between the atezolizumab and placebo groups stratified by NOTCH1 expression (Figure 1C). Importantly, OS among the atezolizumab and placebo treatment groups was similar irrespective of NOTCH2 or REST expression in both the NE and non-NE-enriched subsets (Supplemental Figure 1, A–D; supplemental material available online with this article; https://doi.org/10.1172/JCI185423DS1). We also analyzed long-term survival (LTS; defined as ≥18-month OS) (27) and found a nonsignificant trend toward higher NOTCH1 expression in LTS compared with non-LTS patients in the atezolizumab but not in the placebo arm (Supplemental Figure 2).

High NOTCH1 expression is significantly associated with longer OS with the addition of atezolizumab (anti–PD-L1 inhibitor) to first-line chemotherapy among NE subsets of patients with extensive-stage SCLC in the IMpower133 clinical trial. (A) Unbiased generalized random forest OS analysis comparing atezolizumab with placebo using the 32 genes of the Hallmark Notch signaling gene set within the NE-enriched (NMF1/2/3) subset of the IMpower133 clinical trial. Kaplan-Meier estimates of OS among the atezolizumab and placebo treatment groups of (B) NE-enriched, (C) non-NE-enriched (NMF4), (D) ASCL1-enriched (NMF2/3), and (E) NEUROD1-enriched (NMF1) IMpower133 subsets stratified by high (greater than or equal to median) and low (less than median) NOTCH1 expression. (F) Summary of OS hazard ratios, comparing atezolizumab with placebo based on high NOTCH1 expression among the main IMpower133 subsets. Vertical lines in survival graphs represent censored patients. P values were calculated using a log-rank test. P values were unadjusted, and values less than 0.05 were considered significant. Atezo, atezolizumab; HR, hazard ratio.

We next analyzed the relationship between NOTCH1 expression within the individual NE subsets: ASCL1-enriched (NMF2/3) and NEUROD1-enriched (NMF1). In the ASCL1-enriched subset with high NOTCH1 expression, median OS was nearly doubled with atezolizumab (16.4 months; 95% CI, 10.8–21.6) compared with placebo (8.3 months; 95% CI, 7.5–10.7) (Figure 1D). Strikingly, the HR for death was 0.39 (95% CI, 0.22–0.69; unadjusted P = 0.0012), and the OS rate was more than 3 times higher at 1 year with atezolizumab (61.3%) compared with placebo (17.3%) (Figure 1D). However, in the ASCL1-enriched subset with low NOTCH1 expression, median OS was 2 months shorter with atezolizumab (10.6 months; 95% CI, 7.4–15.9) than with placebo (12.7 months; 95% CI, 10.0–17.3), and the 1-year OS rate was lower with atezolizumab (39.1%) than with placebo (50.6%) (Figure 1D). In the NEUROD1-enriched subset, high NOTCH1 expression was also significantly associated with longer OS with atezolizumab compared with placebo (HR 0.44; 95% CI, 0.21–0.92; unadjusted P = 0.024), whereas low NOTCH1 expression was not (HR 0.79; 95% CI, 0.40–1.55; unadjusted P = 0.49) (Figure 1E). A summary of the relationship between high NOTCH1 expression and OS across the main subsets of IMpower133 is shown in Figure 1F.

Given the differences in survival based on NOTCH1 expression using NMF-defined subsets, we next sought to validate our results using previously defined subsets: Rudin et al. (28) (ASCL1, NEUROD1, POU2F3, YAP1) and Gay et al. (16) (SCLC-A, SCLC-N, SCLC-I, SCLC-P). Among tumors defined by high expression of ASCL1 or NEUROD1 (i.e., NE), high NOTCH1 expression was significantly associated with longer OS with atezolizumab compared with placebo (HR 0.58; 95% CI, 0.38–0.87; unadjusted P = 0.009), whereas low NOTCH1 expression was not (HR 0.90; 95% CI, 0.60–1.34; unadjusted P = 0.60) (Supplemental Figure 3A). Similarly, among SCLC-A and SCLC-N tumors, high NOTCH1 expression was significantly associated with longer OS with atezolizumab compared with placebo (HR 0.52; 95% CI, 0.33–0.83; unadjusted P = 0.005), whereas low NOTCH1 expression was not (HR 1.12; 95% CI, 0.72–1.74; unadjusted P = 0.62) (Supplemental Figure 3B). There were no significant differences in OS between the atezolizumab and placebo groups stratified by NOTCH1 expression among tumors defined by high expression of POU2F3 and YAP1 (i.e., non-NE) or within the SCLC-P subset (Supplemental Figure 4, A and B). Within the SCLC-I subset, we observed prolonged OS with atezolizumab compared with placebo in both low- and high-NOTCH1-expressing tumors (Supplemental Figure 4C). Despite the stark differences in OS between SCLC-I and SCLC-P in the atezolizumab arm (Supplemental Figure 4, B and C), we observed nearly all SCLC-I (82%, n = 40 of 49) and SCLC-P (90%, n = 19 of 21) tumors to have high expression of NOTCH1 (Figure 2A). As MYC has been shown to be a driver of Notch signaling in SCLC (29) and may impair response to ICB in lung cancer (30), we examined MYC expression across these subsets and found very high MYC expression in SCLC-P, but not in SCLC-I or SCLC-A/N (Figure 2B), and no difference in MYC expression between NE-enriched tumors stratified by NOTCH1 expression (Figure 2C). Consequently, after exclusion of SCLC-P tumors, high NOTCH1 expression was associated with significantly longer OS with atezolizumab compared with placebo (HR 0.59; 95% CI, 0.39–0.90; unadjusted P = 0.01) among the remaining IMpower133 dataset, whereas low NOTCH1 expression was not (HR 0.88; 95% CI, 0.59–1.31; unadjusted P = 0.51) (Figure 2D). Importantly, we found no significant association between NOTCH1 expression and OS among NE-enriched (NMF1/2/3) SCLC limited-stage tumors (23, 31) demonstrating that NOTCH1 expression is not prognostic in SCLC (Supplemental Figure 5). Together, our data suggest that NOTCH1 expression is predictive of OS among NE subsets of patients with SCLC treated with first-line ICB plus chemotherapy.

High NOTCH1 expression is significantly associated with longer OS with the addition of atezolizumab to first-line chemotherapy among all extensive-stage SCLC patients in the IMpower133 clinical trial, except those with high-POU2F3-expressing tumors. (A) NOTCH1 expression and (B) MYC expression among IMpower133 subsets defined by Gay et al. (16). (C) MYC expression among high- and low- NOTCH1-expressing tumors in IMpower133, excluding only POU2F3-expressing tumors. (D) Kaplan-Meier estimates of OS stratified by NOTCH1 expression among the atezolizumab and placebo treatment groups of the IMpower133 trial, excluding only POU2F3-expressing tumors. P values were calculated using a log-rank test. P values were unadjusted, and values less than 0.05 were considered significant.

Regulation and expression of NOTCH1 is distinct from those of NOTCH2 and REST in SCLC. Given our data indicating a specific association between NOTCH1 expression, but not NOTCH2 expression, and ICB survival in SCLC, we next sought to elucidate potential differences between NOTCH1 and NOTCH2, as these Notch paralogs have been previously reported to have similar functions in SCLC as tumor suppressors (23) and drivers of NE to non-NE transdifferentiation (24, 25). Using the IMpower133 dataset, we first compared expression of NOTCH1 and NOTCH2 in the NE-enriched (NMF1/2/3) and non-NE-enriched (NMF4) subsets. We found NOTCH2 to be one of the most significantly enriched genes within the non-NE-enriched subset (Figure 3A), along with MYC and REST, as previously reported by Nabet et al. (18). Surprisingly, compared with NOTCH2, NOTCH1 was less upregulated in the non-NE-enriched subset (Figure 3, A and B). NE genes were also less downregulated than expected among NOTCH1-high NE-enriched tumors (Supplemental Figure 6A) compared with the complete downregulation of NE genes evident in our NOTCH1-activated preclinical models (Supplemental Figure 6B). The fraction of high-NOTCH2-expressing tumors in the non-NE-enriched subset was also greater than the fraction of high-NOTCH1-expressing tumors (Figure 3B). To validate these results, we performed differential gene expression analysis between NE-enriched and non-NE-enriched subsets among a combined cohort of limited-stage SCLC tumors (23, 31) and similarly found NOTCH2, but not NOTCH1, to be enriched among the non-NE-enriched subset (excluding POU2F3-high tumors) (Supplemental Figure 6C). We next reanalyzed RNA-Seq data generated from Ireland et al. (29), who showed that Myc activation reprograms NE cell fate through Notch signaling in a SCLC murine model. Upon Myc activation in this model, we observed little to no upregulation of Notch1, whereas Notch2 and Rest were highly upregulated (Figure 3C). Similarly, reanalysis of RNA-Seq data of Rest overexpression in the KP1 SCLC murine cell line (25) showed significant upregulation of Notch2, but not Notch1 (Figure 3D). In sum, these data suggest that NOTCH1 has a distinct pattern of regulation and expression apart from NOTCH2 and REST in SCLC.

NOTCH1 exhibits a regulatory and expression pattern distinct from those of NOTCH2 and REST. (A) Volcano plot showing Notch signaling, NE, and MYC genes differentially expressed between NE-enriched and non-NE-enriched tumors in IMpower133. (B) Stacked box plots showing fraction of patients with high and low NOTCH1 or NOTCH2 tumors among NE-enriched and non-NE-enriched subsets in IMpower133. (C) Reanalysis of RNA-Seq data from Ireland et al. (29) showing expression of Notch1, Notch2, and Rest at multiple time points in RPM cells grown in culture. RPM cells were derived from a Myc-driven SCLC mouse model (Rb1fl/fl;Trp53fl/fl; Lox-Stop-Lox [LSL]-MycT58A). (D) Volcano plot highlighting Notch1 and Notch2 with KP1 Rest overexpression; data from Shue et al. (25).

NOTCH1 reverses silencing of MHC class I and antigen presentation in SCLC. Given the significant association between high NOTCH1 expression and first-line ICB survival, we next assessed for potential mechanisms by which NOTCH1 signaling may mediate immune response by performing gene set enrichment analysis between high- and low-NOTCH1-expressing tumors within the NE-enriched subset of IMpwer133. Using signatures developed to predict pan-cancer response to immunotherapy (32), we found angiogenesis, epithelial-mesenchymal transition (EMT), and protumor cytokines to be the most significantly enriched pathways in high compared with low-NOTCH1-expressing tumors (Figure 4A). We next explored the relationship between NOTCH1 and EMT by performing RNA-Seq across multiple time points in our previously described H82 (NEUROD1) SCLC cell line model, in which HLAs and antigen presentation machinery (APM) genes are upregulated by NOTCH1 intracellular domain (N1ICD) overexpression (20). We found that N1ICD overexpression increased EMT over time in H82 cells (Figure 4B), consistent with a model of EMT as a transitional, rather than binary, process (33). Cell-surface MHC class I expression also increased over time with N1ICD overexpression in concordance with EMT (Figure 4C). To further understand how NOTCH1 signaling might regulate EMT, APM and cell-surface MHC class I expression, we knocked out REST — a downstream Notch signaling gene known to regulate cell fate in SCLC — in H82 cells (24, 25). However, with REST KO and N1ICD overexpression, we did not observe significant differences in EMT by RNA-Seq (Supplemental Figure 7A) or the EMT marker AXL (Figure 4D), nor was there a significant change in cell-surface MHC class I expression (Figure 4E) or APM gene expression (Supplemental Figure 7B). We then directly compared NOTCH1 with REST in driving EMT and APM in SCLC by individually overexpressing N1ICD and REST in H524 (NEUROD1) cells with minimal endogenous expression of either of these proteins. As in H82 cells, long-term overexpression of N1ICD in H524 cells induced EMT and AXL expression, but long-term overexpression of REST did not (Figure 4F and Supplemental Figure 7C). H524 N1ICD-overexpressed cells also had significantly higher cell-surface MHC class I expression (Figure 4G) and higher APM gene expression (Supplemental Figure 7D) than H524 REST overexpressed cells indicating that NOTCH1 was more effective in driving EMT and upregulating antigen presentation than REST. Further supporting these data, N1ICD overexpression in H69 (ASCL1) cells led to significant upregulation of EMT as well as increased cell-surface MHC class I and APM gene expression (Figure 4, H and I, and Supplemental Figure 7, E and F).

NOTCH1 reverses silencing of MHC class I and antigen presentation in SCLC. (A) Gene set enrichment analysis of high- compared with low-NOTCH1-expressing tumors in the NE-enriched subset of IMpower133. (B–E) N1ICD overexpression time course (0 to ≤56 days) in H82 cells with or without REST KO. (B) EMT signature (z scored) at the indicated time points as determined by RNA-Seq. (C) Flow cytometry histograms assessing cell-surface MHC class I expression at the indicated time points. (D) Immunoblot analysis of the indicated proteins. Three single-cell KO clones are shown. (E) Quantification of cell-surface MHC class I expression (data representative of n = 3 independent experiments). (F and G) Long-term (56 days) N1ICD and REST overexpression in H524 cells. (F) Immunoblot analysis of the indicated proteins. (G) Quantification of cell-surface MHC class I expression (data representative of n = 3 independent experiments). (H and I) Long-term (>56 days) overexpression of N1ICD in H69 cells. (H) Immunoblot analysis of the indicated proteins. (I) Flow cytometry assessing cell-surface MHC class I expression (data representative of n = 3 independent experiments). (J and K) Short-term (7 days) and/or long-term (28 days) treatment of COR-L88 cells with DMSO, TAS1440, and TAS1440 plus GSI (BMS-708163, 2 μM) as indicated (data representative of n > 3 independent experiments). (J) Immunoblot analysis of the indicated proteins. (K) Flow cytometry assessing cell-surface MHC class I expression. (L) AXL expression and (M) MHC class I signature (HLA-A, HLA-B, HLA-C, B2M, TAP1, TAP2, TAPBP) stratified by NOTCH1 expression among the NE-enriched subset of IMpower133. (N) Immunoblot analysis of the indicated proteins in H446 suspension, adherent, and H446 adherent N1ICD-overexpressed cells (56 days). (O) Flow cytometry assessing cell-surface MHC class I expression. For flow cytometry graphs, shaded gray histograms represent unstained controls for each condition. Positive cells are shifted to the right of the gray vertical line. P values were calculated using an unpaired 2-tailed Student’s t test. P values less than 0.05 were considered significant.

Given that Notch signaling is dose dependent and N1ICD overexpression may not represent normal physiologic N1ICD levels (34), we next used pharmacologic activation of Notch signaling through LSD1 inhibition (35) as an orthogonal approach to assess the relationship among NOTCH1, EMT, and antigen presentation in SCLC. Consistent with prior reports by Hiatt et al. (36) and Nguyen et al. (37), short-term (7 days) treatment with a potent, reversible LSD1 inhibitor, TAS1440 (Machida et al., manuscript in preparation), broadly activated Notch signaling (i.e., expression of NOTCH1, NOTCH2, and REST) and modestly upregulated cell-surface MHC class I but did not substantially induce AXL in COR-L88 (ASCL1) cells (Figure 4, J and K). Gamma-secretase inhibition (GSI), which has been used to block oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia (29, 35), did not alter the modest upregulation of cell-surface MHC class I with short-term TAS1440 treatment (Figure 4K). In contrast, we observed significant induction of EMT and profound upregulation of surface MHC class I with long-term (28 days) Notch activation (Figure 4, J and K, and Supplemental Figure 7G). Blocking NOTCH1 signaling with concurrent GSI and LSD1 treatment led to partial induction of EMT (Supplemental Figure 7G) and only modest upregulation of cell-surface MHC class I (Figure 4, J and K). Bulk and single-cell RNA-Seq similarly showed strong upregulation of APM gene transcription with long-term Notch activation (Supplemental Figure 7, H and I). MHC class I mass spectrometry analysis demonstrated a significant increase in cell-surface MHC–bound peptides in long-term TAS1440- compared with long-term TAS1440 plus GSI–treated cells (Supplemental Figure 7J). Consistent with our preclinical models, we observed significantly higher expression of AXL and higher expression of MHC class I–related genes among high- compared with low-NOTCH1-expressing NE-enriched tumors in IMpower133 (Figure 4, L and M).

Last, we analyzed expression of NOTCH1, NOTCH2, and REST within NE and non-NE populations of the H446 (NEUROD1) cell line (38, 39) to assess whether these proteins may be coregulated. As expected, we observed little to no expression of NOTCH1, NOTCH2, or REST and high expression of NE proteins in H446 suspension cells (Figure 4N). Interestingly, NOTCH2 and REST, rather than NOTCH1-ICD, were highly expressed in non-NE H446 adherent cells, with low concurrent expression of AXL and cell-surface MHC class I (Figure 4, N and O). Overexpression of N1ICD in the non-NE H446 adherent cells led to upregulation of AXL and cell-surface MHC class I, consistent with our previously described N1ICD overexpression models (Figure 4, N and O). Thus, our results demonstrate that NOTCH1 signaling was a key driver of MHC class I and antigen presentation in SCLC.

Notch signaling drives the immunogenicity of SCLC. Next, we sought to determine whether NOTCH1 could drive antitumor immune response in SCLC. To do this, we treated the well-established KP1 SCLC syngeneic mouse cell line (40–42) long-term ex vivo with and without TAS1440 and TAS1440 plus GSI (Figure 5A). We first measured cell growth after TAS1440 treatment at 7 days and 28 days and found no significant growth inhibition compared with the DMSO-, TAS1440 plus GSI–, and Notch1-KO–treated cells (Supplemental Figure 8, A and B). As in our human SCLC cell line model, long-term KP1 TAS1440–treated cells upregulated Notch signaling, induced EMT based on increased expression of Vim and the cell surface-marker Cd44, increased cell-surface MHC class I, and increased APM gene expression (Supplemental Figure 9, A–C). Blocking active Notch signaling with addition of a GSI to TAS1440 attenuated these observed phenotypes (Supplemental Figure 9, A–C). Given the strong increase in cell-surface MHC class I expression with Notch activation, we assessed whether Notch activation could induce T cell–mediated cytotoxicity by pulsing KP1 cells with OVA peptide (SIINFEKL), then coculturing them with OVA peptide–specific, i.e., OT-I, T cells. TAS1440-treated KP1 cells showed significantly greater cell lysis compared with TAS1440 plus GSI–treated cells (Figure 5B). Moreover, OT-I T cell coculture with TAS1440–treated cells induced greater T cell activation, as evidenced by higher T cell cytokine IFN-γ production, than coculture with TAS1440 plus GSI–treated cells (Figure 5B).

Notch signaling reprograms SCLC tumors from immune-excluded to immune-inflamed through increased T cell infiltration and activation. (A) Schematic of in vitro and in vivo experiments. (B) Percentage lysis and IFN-γ concentration in supernatants of KP1 cells cocultured with OT-I T cells for 3 days after pulsing with OVA peptide. E, effector (OT-I T cells); T, target (KP1 cells) (data representative of n = 3 independent experiments). (C) Tumor growth curves and survival of KP1 allografts in B6129SF1/J immunocompetent and NSG immunocompromised mice (data representative of n = 2 independent experiments). (D–F) Tumor microenvironment analysis of KP1 allograft tumors in B6129SF1/J immunocompetent mice 11 days after subcutaneous inoculation. (D) Flow cytometry assessing tumor T cells. (E) CD3+ and CD8+ T cell IHC. Arrowheads point to T cell clusters. Scale bars: 100 μm. (F) Spatial heatmap of CD3+ T cells analyzed by CODEX. (G) Tumor growth curves of KP1 TAS1440 allografts in B6129SF1/J immunocompetent mice with T cell depletion (upper panel). Isotype, CD4+, and CD8+ T cell depletion (n = 1 independent experiment). Combined CD4+ and CD8+ T cell depletion (n = 2 independent experiments). Flow cytometric analysis confirming T cell depletion in splenocytes (lower panel). P values were calculated using an unpaired 2-tailed Student’s t test or using a log-rank test. P values less than 0.05 were considered significant. Error bars in tumor growth curves (C and G) represent SEM.

We next assessed the immunogenicity of Notch-driven SCLC by subcutaneously inoculating ex vivo treated KP1 cells (DMSO, TAS1440, and TAS1440 plus GSI) into both immunocompromised NSG and immunocompetent B6129SF1/J mice (Figure 5C). All KP1 cells induced tumors in immunocompromised mice. In contrast, tumors formed from TAS1440-treated KP1 cells (hereafter referred to as KP1 TAS1440 tumors) regressed over time in immunocompetent mice. However, tumors formed from DMSO and TAS1440 plus GSI–treated KP1 cells (hereafter referred to as KP1 DMSO and TAS1440 plus GSI tumors) continued to grow (Figure 5C). To validate these results, we repeated this experiment using KP3 cells, another well-validated SCLC syngeneic mouse model (40–42). Like KP1 cells, KP3 TAS1440 cells regressed over time in immunocompetent mice, whereas they induced tumors in immunocompromised mice (Supplemental Figure 9D). KP3 DMSO and TAS1440 plus GSI cells grew in both immunocompetent and immunocompromised mice, but they grew more slowly in immunocompetent mice, suggesting a partial immune response (Supplemental Figure 9D). Overall, these data underscore the role of Notch signaling in regulating SCLC in vivo immunogenicity.

Given these data, we next hypothesized that active Notch signaling may also be an underlying mechanism for in vivo tumor regression of adherent SCLC syngeneic mouse cells, as reported by Mahadevan et al. (17). To test this possibility, we generated adherent KP1 cells (KP1-A cells) by long-term culture, which we confirmed were of the same origin as parental suspension KP1 cells (Supplemental Figure 9E). As expected, KP1-A cells showed strong evidence of EMT (based on high Cd44 cell-surface expression) as well as high MHC class I cell-surface expression (Supplemental Figure 9F). In contrast, concurrent long-term culture of KP1-A cells with a GSI, which blocked Notch1 signaling (Supplemental Figure 9G), hindered upregulation of EMT and cell-surface MHC class I (Supplemental Figure 9F). Crucially, tumors formed from KP1-A cells, but not from KP1-A plus GSI cells, regressed in immunocompetent mice (Supplemental Figure 9H). Taken together, these data demonstrate that Notch signaling is a key mechanism driving in vivo SCLC antitumor immune responses.

Notch signaling reprograms SCLC tumors from immune excluded to immune inflamed through increased T cell infiltration and activation. The robust antitumor immune responses induced by Notch signaling in our SCLC syngeneic mouse models prompted us to evaluate the tumor microenvironment of KP1 DMSO, TAS1440, and TAS1440 plus GSI tumors (Figure 5A). Using flow cytometry, we found significant enrichment of CD4+ and CD8+ T cells in KP1 TAS1440 compared with KP1 TAS1440 plus GSI tumors (Figure 5D). Although there was less robust enrichment of CD8+ compared with CD4+ T cells, KP1 TAS1440 tumors had significantly more activated effector CD8+ T cells than KP1 TAS1440 plus GSI tumors (Figure 5D). Strikingly, KP1 DMSO and KP1 TAS plus GSI tumors were immune excluded, with CD3+ and CD8+ T cells restricted predominantly to the tumor margin, whereas KP1 TAS1440 tumors were immune inflamed, with abundant infiltration of CD3+ and CD8+ T cells within the interior of the tumor (Figure 5E), which was also evident in the KP1-A model (Supplemental Figure 9I). CODEX analysis concordantly revealed a large increase in CD3+ T cell density deep in the tumor core in KP1 TAS1440 tumors compared with KP1 DMSO and TAS1440 plus GSI tumors (Figure 5F).

As the effector functions of CD8+ T cells are known to be supported by the presence of CD4+ T cells (43), we performed in vivo antibody depletion of CD4+ and/or CD8+ T cell subsets in mice with KP1 TAS1440 tumors. Depletion of either CD4+ or CD8+ T cells resulted in tumor growth, whereas isotype-treated KP1 TAS1440 tumors regressed (Figure 5G). Depletion of both T cell subsets led to pronounced tumor growth (Figure 5G), providing evidence that tumor-infiltrating CD4+ and CD8+ T cells both have a critical role in driving antitumor immune responses of Notch-driven SCLC tumors.

Notch1 is critical for the immunogenicity of SCLC. Although GSIs have been used extensively to block Notch signaling in SCLC (29, 35), these drugs have also been shown to target other membrane proteins (44, 45). Therefore, to assess the specific relationship between Notch1 and antitumor immune response in SCLC, we knocked out Notch1 in KP1 cells and treated these cells long-term ex vivo with TAS1440 (Figure 6A). Despite similarly high expression of Notch2 and Rest and downregulation of NE proteins (Figure 6A), KP1 TAS1440 Notch1-KO cells had lower cell-surface MHC class I expression and decreased EMT, as evidenced by lower Vim and cell-surface Cd44 expression compared with TAS1440-treated Notch1 WT cells (Figure 6B). Consistent with these findings, OT-I T cell killing assays demonstrated reduced cytotoxicity against KP1 Notch1-KO cells compared with Notch1 WT cells following TAS1440 treatment, further supporting a critical role for Notch1 in enhancing antigen presentation and T cell–mediated killing (Figure 6C). Moreover, in immunocompetent mice, KP1 TAS1440 Notch1-KO cells induced tumor growth, whereas tumors induced from Notch1 WT cells regressed (Figure 6D). Using flow cytometry, we found significant depletion of both total CD8+ T cells and activated CD8+ T cells in KP1 TAS1440 Notch1-KO tumors compared with Notch1 WT tumors (Figure 6E). Moreover, tumors formed from KP1 cells with N1icd overexpression (Supplemental Figure 9J) regressed over time in immunocompetent mice, whereas such tumors grew in immunocompromised mice (Figure 6F). These data demonstrate that Notch1 was required to reverse silencing of antigen presentation and induce a robust CD8+ T cell–mediated response in SCLC. Concordantly, we observed significant enrichment of a T cell signature (32) in high- compared with low-NOTCH1-expressing NE-enriched tumors in IMpower133 (Figure 6G).

Notch1 is the critical driver of the immunogenicity of SCLC. (A–E) KP1 SCLC mouse cells with or without Notch1 KO treated long-term (>28 days) with TAS1440. (A) Immunoblot analysis of Notch signaling, NE, and EMT proteins. (B) Flow cytometry histograms assessing cell-surface H2 and Cd44 expression. Shaded gray histograms represent unstained controls for each condition. Positive cells have an H2 or Cd44 signal higher than the referenced gray vertical line. Data representative of n = 3 independent experiments. (C) T cell–mediated killing assay showing remaining tumor cells assessed by crystal violet staining after coculture of KP1 cells with OT-I T cells for 3 days following OVA peptide pulsing. E, effector (OT-I T cells); T, target (KP1 cells). Colony area for each E:T condition was quantified and normalized to the no–T cell control (E:T = 0) within each group. (D) Tumor growth curves of KP1 TAS1440 allografts in B6129SF1/J immunocompetent mice and (E) flow cytometry T cell analysis 11 days after subcutaneous inoculation. (F) Notch1-icd overexpression in KP1 cells treated with doxycycline ex vivo long-term (>28 days) before subcutaneous inoculation into mice. Tumor growth curves of KP1 mN1icd allografts in immunocompetent and immunocompromised mice. (G) T cells signature stratified by NOTCH1 expression among NE-enriched tumors in IMpower133. Error bars in tumor growth curves (D and F) represent SEM. P values were calculated using an unpaired 2-tailed Student’s t test. P values less than 0.05 were considered significant.

NOTCH1 reverses silencing of antigen presentation in SCLC through reactivation of STING. We next sought to decipher potential mechanism(s) by which NOTCH1 reverses immune suppression in SCLC by performing bulk RNA-Seq and gene set enrichment analysis (GSEA) between TAS1440- and TAS1440 plus GSI–treated COR-L88 and KP1 cells. The immune system gene set was a top differentially enriched pathway, with interferon-inducible genes highly upregulated in TAS1440- compared with TAS1440 plus GSI–treated cells (Supplemental Figure 10A). Findings were similar in H82 cells with and without N1ICD overexpression (Supplemental Figure 10B). We therefore postulated that expression of STING, a known regulator of interferon and cytokine production (46), may be higher in NOTCH1-driven cells. Indeed, we observed upregulation of STING in long-term (28 days) NOTCH1-driven COR-L88 cells (Figure 7A and Supplemental Figure 10C) but minimal STING upregulation in short-term (7 days) NOTCH1-driven COR-L88 cells (Figure 7A). STING1 expression increased concurrently over time with EMT in H82 cells with N1ICD overexpression with or without REST KO (Figure 7B and Supplemental Figure 10D). We also observed upregulation of STING after N1ICD overexpression in non-NE H446 adherent cells (Figure 7C) and lower expression of Sting in KP1 TAS1440 Notch1-KO compared with WT cells (Figure 7D). Reanalysis of RNA-Seq data from Hong et al. (47) similarly showed low Sting1 expression among murine SCLC tumors with Notch1 KO (N1_Mutant_c188) in contrast to Notch2-KO tumors (N2_Mutant_cK60 and cK62) (Figure 7E). To further investigate potential differences between NOTCH1 and NOTCH2, we overexpressed N1ICD and N2ICD in COR-L88 cells (Supplemental Figure 10E) and found that N1ICD overexpression led to more robust STING protein upregulation than did N2ICD overexpression (Figure 7F). Additionally, N1ICD overexpression upregulated the EMT marker VIM to a greater extent than N2ICD overexpression, suggesting distinct roles for NOTCH1 and NOTCH2 in regulating EMT and tumor-intrinsic STING expression (Supplemental Figure 10F). Importantly, there was significantly higher STING1 expression in high- compared with low-NOTCH1-expressing NE-enriched tumors in IMpower133 (Figure 7G).