Maximum grade adverse events, regardless of attribution. AEs are summarized as n (%) for each maximum grade.

Consort diagram for status of trial. Progression (PD) includes protocol defined progression and clinical/symptomatic progression per physician.

Immune cell functional states differ in clinical responders compared to non-responders. (A) Heat maps summarizing log2 fold changes resulting from statistical scaffold analysis of functional markers 4-1BB, CD44, CTLA-4, GITR, HLA-DR, ICOS, Ki-67, OX40, PD-1, PD-L1, PD-L2, TIGIT, TIM3 and VISTA are shown for Week 1, Week 5, and Week 9, when comparing responders to non-responders as defined by rPFS.

Immunofluorescence staining of PD-L1+ CD68 myeloid cells in the bone biopsies. A. RNAscope staining of DAPI (cyan), CD68 (orange), PD-L1 (magenta), Tim3 (green), TIGIT (white) and overlay of bone marrow metastasis. CD68+ cells that co-express PD-L1 are indicated with arrow. Tim3+ cells are indicated with pointer. Scale bar, 20 μm. B. Scatterplot of PDL1+ CD68+ cell frequencies of tissue imaging data and PDL1+ CD14+ CD3-CD19- myeloid cell frequencies of CyTOF data from blood samples taken at the same timepoint (baseline, n=10 patients). R2 value is based on Pearson correlation. C. Scatterplot of TIM3+ CD68- cell frequencies on tissue imaging and TIM3+ CD3+ T cell frequencies of CyTOF data from blood samples taken at the same timepoint (baseline, n=10 patients). R2 value is based on Pearson correlation.

Kaplan-Meier estimates of time to treatment failure (TTF), radiographic progression-free survival (rPFS), and overall survival (OS) by A. baseline CD4+FoxP3- (effector) proportion above (high) vs. below (low) median. B. baseline CD8+ proportion above vs. below median. C. baseline CD8+ / CD4+FoxP3+ (Treg) ratio above vs. below median. N.B. Patients were event-free for 2 months as they need to have on treatment biopsy at week 8 of treatment.

Time to event (months) clinical endpoints by baseline and change in CD4+/FoxP3- (effector) proportion, CD8+ proportion, CD8+ / CD4+FoxP3- (Treg) ratio

Changes in A. CD4+FoxP3- effector cells and B. ratio of CD8+ to CD4+/FoxP3+ (Treg) cells from baseline to 8 weeks on treatment

Proportion of patients who experienced any or given category of symptomatic skeletal events by the treatment arms

Kaplan-Meier estimates of time to treatment failure (TTF), radiographic progression-free survival (rPFS), and overall survival (OS) by A. CD4+FoxP3- (effector) proportion increase vs. decrease from baseline to 8-week biopsy. B. CD8+ increase vs. decrease. C. CD8+ / CD4+FoxP3+ (Treg) ratio increase vs. decrease. N.B. Patients were event-free for 2 months as they need to have on treatment biopsy at week 8 of treatment.

Clinical characteristics at time of randomization of PSA responders vs. non-responders

Swimmer’s plot of treatments received. Number of radium-223 and pembrolizumab doses received are listed to the left and right of the bars, respectively. The brackets “[” and “]” represent the planned break period from radium-223 dosing with “[” representing cycle 3 day 28 and “]” representing cycle 4 day 1 when the interval between these was greater than 28 days. Patients could resume radium treatment at radiographic progression by PCWG2 criteria (“△”) if there were no new visceral metastases, but two patients (bars 7 and 13 below) resumed without confirmed radiographic progression.

Summary of genetic findings from targeted sequencing of ctDNA and whole exome sequencing (WES) from bone biopsy specimen.

Radium-223 was administered as 55 kBq/kg q4weeks and pembrolizumab as 200 mg q3weeks. Biopsies were performed pre-treatment and after 8 weeks of treatment.

Docetaxel is the first-line chemotherapy for metastatic prostate cancer (PC), but clinically meaningful mechanisms of resistance remain to be established. Here we show, in an in vivo model of docetaxel resistant PC patient-derived xenografts, increased expression of genes that drive development of multiciliated cells including FOXJ1 and its effectors, many of which regulate microtubules (MTs). Mechanistically, FOXJ1 overexpression confers docetaxel resistance in vitro and in vivo, which is associated with decreased docetaxel-mediated MT bundling. Overexpression of a MT-associated FOXJ1-regulated gene (TPPP3) has similar effects. Conversely, FOXJ1 knockdown impairs basal MT function, enhances taxane binding to MTs, and increases docetaxel sensitivity. These results establish mechanistic causality between the FOXJ1 signaling axis, MT biology, and taxane resistance. Clinically, FOXJ1 gene amplification is increased in taxane-treated PC patients. Moreover, in the CHAARTED clinical trial of docetaxel combined with androgen deprivation for metastatic PC, higher baseline FOXJ1 is predictive of decreased survival in PC patients treated with docetaxel, further supporting clinical relevance. Together, these findings identify a previously unrecognized clinically impactful mechanism of taxane resistance whose exploitation could stratify patients who will not benefit from taxane treatment.

437 Background: CheckMate 214 established nivolumab and ipilimumab (NIVO+IPI) as a first line standard of care regimen with superior survival and durable response versus sunitinib (SUN) in advanced RCC. Previous studies have shown that high levels of circulating KIM-1 are associated with worse prognosis and reduction in KIM-1 levels is associated with benefit from adjuvant immunotherapy. In this post-hoc analysis we evaluated whether KIM-1 levels at baseline and after 1 cycle of NIVO+IPI or SUN are associated with treatment outcomes in CheckMate 214. Methods: Patients with advanced RCC were randomized to NIVO+IPI or SUN as previously described. Serum KIM-1 was measured at baseline and 3 wks after first treatment dose using an enzyme based electrochemiluminescence assay. The association between KIM-1 levels and clinical outcomes was evaluated using Kaplan-Meier and Cox proportional hazards analyses. Results: We analyzed serum from 821 patients (75% of the CM 214 ITT population). Median KIM-1 at baseline was 660.4 pg/mL. Across both arms, higher KIM-1 levels were associated with shorter overall survival (OS) independent of IMDC risk group, nephrectomy status, and tumor burden. Benefit for NIVO+IPI versus SUN was seen across KIM-1 tertiles. Decrease in KIM-1 from baseline to C2D1 was strongly associated with progression free survival (PFS) and OS among patients treated with NIVO+IPI (Median PFS 70.8 months vs 4.2 months for patients with >30% decrease vs >30% increase in KIM-1, with median OS 85.4 vs 26.6 months, overall response rate (ORR) 69.3 % vs 13.9%), but not in patients treated with SUN. Conclusions: In CheckMate 214, increased levels of baseline circulating KIM-1 were associated with worse clinical outcomes both in NIVO+IPI and SUN arms. The extent of reduction in serum KIM-1 just 3 wks after single cycle of NIVO+IPI was associated with long term efficacy of this IO doublet. Circulating KIM-1 may be a useful minimally invasive biomarker for monitoring patients on RCC immunotherapy. KIM-1 change at 3 weeks (prior to second dose of NIVO+IPI) and association with outcomes (NIVO+IPI arm). 3 week KIM-1 change N (%) ORR, % (95% CI) mPFS, months (95% CI) mOS, months (95% CI) >30% Decrease 140 (31.7) 69.29 (60.94-76.80) 70.80 (17.84- NA) 85.36 (63.08- NA) >10-30% Decrease 87 (19.7) 36.78 (26.69-47.80) 11.43 (6.28-18.20) 66.14 (40.44-80.10) <10% Change 86 (19.5) 30.23 (20.79-41.08) 15.41 (10.32-20.73) 52.70 (30.26-70.67) >10-30% Increase 56 (12.7) 23.21 (12.98-36.42) 7.13 (4.17-16.79) 40.34 (23.82-58.41) >30% Increase 72 (16.3) 13.89 (6.87-24.06) 4.17 (3.02-8.08) 26.61 (18.79-38.44)

Docetaxel is the first-line chemotherapy for metastatic castration-resistant prostate cancer, but clinically meaningful mechanisms of resistance remain to be established. We generated an in vivo model of docetaxel resistance using castration-resistant patient-derived xenografts and found increased expression of genes that drive development of multiciliated cells, including FOXJ1 and its effector genes, many of which regulate ciliary microtubules. Mechanistically, FOXJ1 overexpression conferred docetaxel resistance in vitro and in vivo, which was associated with decreased docetaxel-mediated microtubule bundling. Overexpression of a microtubule-associated FOXJ1-regulated gene (TPPP3) had similar effects. Conversely, FOXJ1 knockdown impaired basal microtubule function, enhanced taxane binding to microtubules, and increased docetaxel sensitivity. These results establish mechanistic causality between the FOXJ1 signaling axis, microtubule biology, and taxane resistance. Clinically, FOXJ1 gene amplification was increased in taxane-treated prostate cancer patients. Moreover, in the CHAARTED clinical trial of docetaxel combined with androgen deprivation for metastatic prostate cancer, higher baseline FOXJ1 was predictive of decreased survival in prostate cancer patients treated with docetaxel, further supporting clinical relevance. Together these findings identify a previously unrecognized clinically impactful mechanism of taxane resistance whose exploitation could stratify patients that will not benefit from taxane treatment. Ada Gjyrezi, Fang Xie, Daniel E. Fein, Maryam Labaf, Larysa Poluben, Olga Voznesensky, Aniket Gad, Eva Corey, Rupal S. Bhatt, Steven Balk, Paraskevi Giannakakou. The multiciliated transcription factor FOXJ1 modulates microtubule dynamics and mediates taxane resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3081.

Abstract Introduction: CheckMate 914 (CM-914) Part A is a double-blind, phase III randomized trial of the Nivolumab (NIVO) plus Ipilimumab (IPI) vs placebo (PBO) in localized ccRCC. Our prior report from this study suggested a disease-free survival (DFS) benefit for NIVO+IPI among patients with Fuhrman grade 4, TNM stages PT2a and PT4, or sarcomatoid features, although the sample size was limited. It is known that high circulating KIM-1 is associated with worse DFS after nephrectomy. In this exploratory post hoc analysis, we investigated whether high KIM-1 may help identify a subset of patients who benefit from adjuvant NIVO+IPI. Methods: Patients (n=816) with RCC after nephrectomy were randomized in CM-914 Part A to receive NIVO+IPI or PBO as previously described. Assessment of KIM-1 levels was performed using enzyme linked immunoassay (ELISA) on pre-treatment (n=584) and matched on-treatment blood samples (n=584). We used pre-treatment tumor samples to assess PD-L1% tumor cell expression (%TC) in an PD-L1 IHC 28-8 pharm Dx assay. The association between biomarkers and DFS outcomes was investigated by Kaplan-Meier (KM) and Cox proportional hazards analysis. Results: Median baseline serum KIM-1 level was 102 (9.9 - 1055.7) pg/mL. Serum KIM-1 levels were higher in males vs females, ≥65 yrs vs <65 yrs, Asian vs white patients, and patients with partial vs radical nephrectomy. In the PBO arm, subjects with highest quartile of pre-treatment KIM-1 had significantly worse DFS than those from the three lower quartiles. In contrast, this DFS risk among the subjects within the highest KIM-1 quartile was mitigated with NIVO+IPI treatment. Among patients within the highest quartile of pre-treatment KIM-1, there was trend for better DFS for NIVO+IPI versus PBO, HR=0.6 (0.34-1.04). Increase in KIM-1 during study therapy was positively associated with higher DFS rate in both arms. Multivariable analysis showed that PD-L1 %TC was predictive at predefined PD-L1 cutoffs (>=1%, >=5%, and >=10%), associating with improved DFS compared to placebo. Subjects with high PD-L1 expression had a DFS benefit from NIVO+IPI independent of KIM-1. Conclusion: Circulating KIM-1 and tumor PD-L1 expression may enrich for benefit from IO therapy in adjuvant ccRCC and hence holds promise for informing risk stratification and patient inclusion in neoadjuvant or adjuvant clinical trials. Citation Format: Sai Vikram Vemula, Wenxin Xu, Yu Wang, Xiaowen Liu, Jorge Ruiz de Somocurcio, David McDermott, Jun Li, Rupal Bhatt, Chung-Wei Lee, Burcin Simsek, Saurabh Gupta, Robert Motzer. High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5151.

Abstract The checkpoint immunotherapeutic pembrolizumab induces responses in a small minority of patients with metastatic castration-resistant prostate cancer (mCRPC). Radium-223 (R223) may increase immunogenicity of bone metastases and increase pembrolizumab (P) activity. In a randomized phase II study, we assessed the effect of R223+P compared with R223 on tumor immune infiltration, safety, and clinical outcomes in patients with mCRPC. The primary endpoint was differences in CD4+ and CD8+ T-cell infiltrate in 8-week versus baseline bone metastasis biopsies; secondary endpoints were safety, radiographic progression-free survival (rPFS), and overall survival (OS). Of the 42 treated patients (29 R223+P, 13 R223), 18 R223+P and 8 R223 patients had evaluable paired tumor biopsies. Median fold-change of CD4+ T cells was −0.7 (range: −9.3 to 4.7) with R223+P and 0.1 (−11.1 to 3.7) with R223 (P = 0.66); for CD8+ T cells, median fold-change was −0.6 (−7.4 to 5.3) with R223+P and −1.3 (−3.1 to 4.8) with R223 (P = 0.66). Median rPFS and OS was 6.1 (95% confidence interval: 2.7–11.0) and 16.9 months [12.7–not reached (NR)], respectively, with R223+P and 5.7 (2.6–NR) and 16.0 (9.0–NR), respectively, with R223. Although R223+P was well tolerated with no unexpected toxicity, the combination did not improve efficacy. High-dimensional flow cytometry demonstrated minimal immune modulation with R223, whereas R223+P induced CTLA-4 expression on circulating CD4+ T cells. Clinical responders possessed lower circulating frequencies of Ki67+ T and myeloid cells at baseline and higher circulating frequencies of TIM-3+ T and myeloid cells by week 9. Although R223+P did not induce T-cell infiltration into the tumor microenvironment, exhaustion of induced peripheral T-cell immune responses may dampen the combination's clinical activity.