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8501 Background: TROPION-Lung02 (NCT04526691) evaluated the TROP2-directed antibody-drug conjugate (ADC) Dato-DXd plus pembro combination with or without Pt-CT in aNSCLC. Here we report primary analyses of pts receiving combination therapy in the 1L setting. Methods: Pts across 6 cohorts were dosed with Dato-DXd (4 or 6 mg/kg) plus pembro 200 mg alone (doublet) or with pembro plus Pt-CT (triplet; cisplatin 75 mg/m 2 or carboplatin AUC 5) Q3W. PD-L1 expression (tumor proportion score) was assessed locally by immunohistochemistry (22C3 assay). Primary objectives were safety and tolerability; efficacy was a secondary objective. Results: As of Apr 29, 2024, 96 pts received either the doublet (n=42) or triplet (n=54) combination as 1L therapy; 29% and 15% of pts were ongoing, respectively. Median ages were 65 (doublet) and 64 years (triplet). Median treatment durations were 9.7 and 5.8 months, respectively. Stomatitis (doublet, 57%; triplet, 33%) and nausea (doublet, 42%; triplet, 48%), primarily Gr 1–2, were the most common adverse events (AEs) across both regimens. Treatment related serious AEs occurred in 5 (12%) and 12 (22%) pts in each cohort and no deaths related to study drug were seen. Efficacy outcomes, including by histology, are summarized in the Table. Biomarker analyses, including efficacy by PD-L1 status, will be presented. Conclusions: In this largest data set to date evaluating an ADC combined with an anti-PD-1/L1 agent in the 1L setting, the combination of Dato-DXd plus pembro treatment both with and without Pt-CT elicited durable antitumor activity in pts with aNSCLC. Tolerability of the combinations was as expected, based on known profiles of the individual agents. Clinical trial information: NCT04526691 . All 1L (n=96) 1L, Nonsquamous (n=75) 1L, Squamous (n=21) Response, n (%) Doublet (n=42) Triplet (n=54) Doublet (n=33) Triplet (n=42) Doublet (n=9) Triplet (n=12) Confirmed objective response rate 23 (55) 30 (56) 17 (52) 24 (57) 6 (67) 6 (50) Complete response 1 (2) 2 (4) 1 (3) 2 (5) 0 0 Partial response 22 (52) 28 (52) 16 (49) 22 (52) 6 (67) 6 (50) Stable disease 14 (33) 18 (33) 12 (36) 14 (33) 2 (22) 4 (33) Progressive disease 3 (7) 2 (4) 3 (9) 1 (2) 0 1 (8) Disease control rate a 37 (88) 48 (89) 29 (88) 38 (91) 8 (89) 10 (83) Median duration of response, mo (95% CI) 20.1 (9.7–NE) 13.7 (5.7–NE) 24.9 (9.7–NE) 18.0 (8.0–NE) 12.0 (5.5–NE) 5.5 (4.1–NE) Median PFS, mo (95% CI) 11.2 b (8.2–21.3) 6.8 c (5.5–11.1) 11.2 (6.1–21.3) 10.8 (5.5–17.3) 10.2 (0.4–NE) 6.7 (1.0–8.2) a Proportion of pts with confirmed CR + PR + SD at 12 wks. b.c Median (95% CI) PFS follow-up, mo: b 17.3 (11.3−26.8); c 23.5 (17.3−27.9). mo, months; NE, not evaluable.

604 Background: The addition of OFS to standard endocrine therapy (ET) improves iDFS for preM women with early-stage hormone receptor positive (HR+) breast cancer (BC). Incomplete OFS occurs in a subset of women, the long-term clinical significance of which is unclear. This study evaluates local sE2 levels in preM women receiving OFS with an oral SERD in the neoadjuvant (NA) setting. Methods: The EOP is a I-SPY2 sub-study investigating NA endocrine-based strategies in pts with HR+ HER2- Stage 2/3 BC predicted to have lower benefit from chemotherapy. Pts were randomized to oral amcenestrant 200 mg/d, alone or in combination with either letrozole 2.5 mg/d or abemaciclib 150 mg bid. Additional pts were randomized to a standard of care (SOC) arm consisting of an aromatase inhibitor. All PreM pts received OFS with monthly GnRHa up to 2 wks prior to C1D1 of study therapy. Pts were treated for 6 months prior to surgery. sE2 levels were collected locally prior to each GnRHa injection. Tumor Ki67 was assessed at 3 weeks and surgery, and associated with sE2 level. Statistical significance was determined using a two-sided Student’s t-test and a significance level 0.05. Results: Between 5/2021-8/2022, 74 pts were enrolled to an amcenestrant containing arm, 40 of whom were preM. 38/40 pts had at least one local sE2 level measured in follow up. Of these 38 pts (median age 45 years), 37 suppressed sE2 into the postmenopausal (postM) range at one or more follow-up timepoints. Of the 37 pts that completely suppressed into the postM range, 6 pts had sE2 levels rebound into the preM range (mean 319 pg/mL, range 20-848 pg/mL) with peak sE2 levels occurring at a median of 12 weeks from C1D1. One pt never suppressed to the postM range (peak sE2 1227 pg/mL) and was found to have an ovarian cyst after 3 months on amcenestrant, requiring surgery. One additional pt had multiple ovarian cysts (sE2 84 pg/mL). Median age of the 7 pts who had sE2 rebound was 43 years. There was no significant difference in Ki67 at 3 weeks and surgery between pts whose sE2 levels remained suppressed (median Ki67 2.0%) compared to those whose estradiol rebounded into the preM range or never suppressed (median Ki67 1.5%). In the 5 pts whose sE2 rebounded to > 200 pg/mL, all pts had tumor Ki67 < 10% at 3 weeks and surgery. Between 12/2022 and 8/2023, 20 pts were enrolled to AI +/- OFS and 11 pts were preM. Of the 11 preM pts, all pts suppressed sE2 to the postM range. No SOC patients had sE2 levels rebound into the preM range. Conclusions: In this study of neoadjuvant oral SERD with monthly OFS, 7/38 (18.4%) preM pts had sE2 levels remain or rebound into the preM range. sE2 levels did not appear to impact Ki67 suppression on NA ET. Work up for ovarian cysts should be considered in pts with symptoms, or significantly elevated or persistent sE2 levels. Clinical trial information: NCT01042379 .

612 Background: EOP, an I-SPY2 sub-study, evaluates the tolerability and activity of novel endocrine strategies in stage 2/3 breast cancer (BC) patients (pts) predicted to have lower chemotherapy benefit. Laso, a selective estrogen receptor modulator (SERM), has shown favorable toxicity profile and activity in HR+/HER2- endocrine-resistant metastatic BC. Methods: Pts with Stage 2/3 HR+/HER2-, MammaPrint (MP) low risk BC were enrolled. Pts with MP High1 BC were included if clinically node-negative. Pts received oral laso 5 mg daily for six 28-day cycles. Laso was continued until the day prior to surgery. Premenopausal pts received ovarian function suppression (OFS) starting C2D1. The primary endpoint was feasibility (>75% of patients completing >75% study therapy). Baseline (T0), 3-wk (T1) biopsies, and the surgical specimen (T3) was assessed centrally for Ki-67. Breast MRI functional tumor volume (FTV) was performed at T0, T1, 12 weeks (T2), and pre-operatively (T3). Blood was collected for tumor informed ctDNA at T0, T1, T2, T3. Advance event (AE) was assessed using CTCAE V5. Results: From 3/2023 to 5/2024, 20 pts were enrolled. Median age 50.5 years, 50% premenopausal, and 1 male pt. 60% cN0, 80% MP low-risk signature. 18 (90%) pts completed >75% study therapy. Two pts discontinued treatment due to pt preference. Median Ki67 at T0 was 14.7%. At T1, 87.5% of pts remained or suppressed Ki67 to <10% and 37.5% suppressed to <2.7%. Ki67 at T1 was similar between pre-and postmenopausal pts despite OFS (Table). The median MRI FTV was 8.4 cc at T0, and 3.4 cc at T3. Median % FTV reduction from T0 to T3 was -47.5%. 2/20 pts (10%) achieved a modified PEPI score of 0. No patients achieved completed pathological response. Of the 16 pts with RCB results, 2 (12.5%) RCB-1, 6 (37.5%) RCB-2, 8 (50%) RCB-3 disease. 14 pts had ctDNA available at T0. 4/14 were ctDNA+ at T0, 2 of whom became ctDNA negative, and 2 remained ctDNA+. 10/14 pts were ctDNA negative at T0, 8 of whom remained negative, 2 became positive at T1 then cleared. All AEs were grade(G) 1 except 1 pt with G2 hot flashes. Most common AEs include hot flashes (85%), constipation (50%), fatigue (50%), and nausea (35%). One pt had G3 hypersensitivity and hypertension, both unrelated to therapy. Conclusions: Neoadjuvant laso demonstrates a favorable AE profile and promising anti-tumor activity in suppressing 3-wk Ki67 and MRI FTV change in pts with HR+ HER2-negative early BC. Ki67 suppression in premenopausal pts was seen in the absence of OFS. Clinical trial information: NCT01042379 . Ki67 expression at pre-treatment, and 3-wk time point. All Patients(n=20) Premenopausal (n=10) Postmenopausal (n=9) Median Ki67 expression Baseline 10.0% 12.5% 10.0% 3-wk 5.1% 3.0% 6.0% Number of pts with Ki67 expression <10% at 3-wk 87.5% 1 87.5% 85.7% Number of pts with Ki67 expression <2.7% at 3-wk 37.5% 50% 28.6% 1 Include the one male pt.

504 Background: Axillary surgery in breast cancer is used for staging and therapeutic purposes, but axillary lymph node dissection (ALND) confers high risk of complications including lymphedema. Accordingly, trials have focused on right-sizing axillary surgery, with options ranging from complete omission of surgery, sentinel lymph node (SLN) surgery, targeted axillary dissection (TAD), or ALND. Identifying a biomarker to reliably predict nodal burden would facilitate accurate selection of surgical management. We evaluated whether the presence or absence of ctDNA in the blood pre and post NAC is associated with residual nodal burden. Methods: I-SPY2 is a prospective, multicenter NAC trial for patients with clinical stage II-III high-risk breast cancer. Patients are randomized to novel NAC agents, with pathologic complete response being the primary endpoint. As part of the trial, serial ctDNA is assessed with a highly sensitive tumor-informed assay using up to 16 patient-specific tumor mutations (Signatera) at baseline, 3 weeks, 12 weeks, and post-NAC. We determined whether ctDNA positivity or negativity post-NAC, and the change in ctDNA status baseline/post-NAC (-/-, -/+, +/+, +/-) are associated with ypN category (N0, N1, N2). Results: ctDNA status was available post-NAC in 495 patients and change in ctDNA status from baseline was available in 493. At baseline, ctDNA was detected in 160/220 (72.3%) cN0 patients and 227/273 (83.2%) cN+ patients (p=0.006). Post-NAC, ctDNA was detected in 11/220 (5%) cN0 patients and 34/273 (12.5%) cN+ patients (p=0.004). While baseline ctDNA status was not associated with ypN category, there was a significant association between post-NAC ctDNA status and ypN category. For ctDNA + patients post-NAC, 33.3% were ypN0, 31.1% were ypN1, and 35.6% were ypN2 at surgery; in contrast, for ctDNA - patients post-NAC, 67.1% were ypN0, 23.1% were ypN1, and 9.8% were ypN2 (p<0.0001). Dynamic ctDNA changes were also associated with ypN category, with significantly more ypN0 patients among ctDNA -/- or ctDNA +/- cases, and more ypN2 patients in those who did not clear ctDNA (+/+) (p=0.0001, Table). Conclusions: To our knowledge this is the first study to demonstrate a significant relationship between ctDNA and ypN category, which has important surgical implications. CtDNA negativity was associated with low likelihood of ypN2 disease, making these patients excellent candidates for SLN surgery or TAD. Ongoing analyses will incorporate receptor subtype and timing of clearance of ctDNA. CtDNA in breast cancer may help tailor surgical management of the axilla, potentially reducing patient morbidity without compromising prognostic information. ctDNA baseline/post-NAC ypN0 (n=317) ypN1 (n=116) ypN2 (n=60) ctDNA -/- (n=105) 72.4% 19.1% 8.6% ctDNA -/+ (n=1) 0.0% 100.0% 0.0% ctDNA +/+ (n=44) 34.1% 29.6% 36.4% ctDNA +/- (n=343) 65.9% 23.9% 10.2%

3008 Background: Numerous studies have demonstrated the prognostic value of ctDNA analysis after neoadjuvant chemotherapy for early-stage high-risk breast cancer. Few studies have characterized ctDNA in pts receiving NET for early-stage hormone receptor positive (HR+)/HER2- BC that is predicted to benefit less from chemotherapy. Methods: Cell-free DNA (cfDNA) was isolated from 432 plasma samples from 108 pts enrolled in the I-SPY2 EOP trial. Pts had Stage 2/3 HR+/HER2-, MammaPrint low or high risk 1 BC. Pts were randomized to one of 7 neoadjuvant-based treatment arms including arms containing AI, Z-endoxifen, Lasofoxifene, vepdegestrant (ARV-471), and Abemaciclib. Pts were treated for 6 months prior to surgery. Blood was collected at baseline (T0), 3 weeks (T1), 12 weeks (T2), and 6 months (T3). A personalized ctDNA test (Signatera) was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The chi-square test was used to assess associations between categorical variables, and the Wilcoxon rank-sum test was used to evaluate differences in medians. Results: ctDNA information was available for 101 patients at baseline (T0) (Table 1). At T0, 36 (35.6%) patients were ctDNA-positive. 23/36 (63.9%) became ctDNA-negative and 13/36 (36.1%) remained ctDNA-positive. At T0, 65/101 patients (64.4%) were ctDNA-negative. Of these, 57 (87.7%) remained negative, while 8 (12.3%) became ctDNA-positive at T1 before reverting to ctDNA-negative. A higher percentage of ctDNA-positive patients at T0 were cN+ compared to ctDNA-negative pts (p = 0.036, 64% vs. 40%). Additionally, ctDNA-positivity at T0 was strongly associated with higher Ki67 (p = 0.03) and larger functional tumor volume by MRI at baseline (p = 0.03). T3/T4 and high-grade tumors at baseline were also associated with having ctDNA positivity at baseline, though this was not statistically significant (p = 0.34 and 0.058, respectively). Conclusions: In this study of pts with Stage 2/3 HR+ HER2- BC with largely MammaPrint low risk signatures, over one-third of pts had detectable ctDNA at baseline. Detectable ctDNA at baseline was associated with cN+ disease, larger FTV, and high baseline Ki67. The majority of pts with positive ctDNA at baseline cleared the ctDNA on NET. Clinical trial information: NCT01042379 . Clinicopathologic characteristics of EOP patients. All Patients(N=101) Age at screening 55 (27-80) years* Clinical N Stage Node+ (cN+) 49 (48.5%) Node- (cN-) 52 (51.5%) SET status High 84 (83.2%) Low 14 (13.9%) Missing 3 (2.97%) MammaPrint (MP) risk** High risk 1 (H1) 15 (14.9%) Low risk 86 (85.1%) *Mean(min-max). **H1: MP score between 0 and -0.57; low: MP score between 0 and 0.355.

1015 Background: T-DXd is an antibody-drug conjugate approved for advanced HER2+/low/ultra-low breast cancer and multiple other solid tumors. T-DXd carries a rare but serious risk of ILD (incidence 12-15%), requiring frequent imaging and symptom evaluation. For > grade (G) 2 ILD, guidelines recommend permanent drug discontinuation. For asymptomatic G1 ILD, drug is held with the option for rechallenge (RC) if imaging findings resolve. Limited data exist on outcomes of RC after ILD in diverse real-world patients (pts). Methods: In this multi-center retrospective study, we analyzed pts with T-DXd related ILD treated from 2017-2024. Pts with ILD were identified via chart/ICD code review. Adjudication of T-DXd related ILD was based on treating providers’ assessment and graded via CTCAE v5. We collected pt demographics, T-DXd and steroid dosing, imaging results, and outcomes after RC. Statistical analysis was performed using Wilcoxon rank sum and Fisher’s exact tests. Results: Four centers treated 712 pts with T-DXd, with a 9.1% rate of any grade ILD (n=65). One other center reported only RC data in 18 pts with ILD. In total, 47 pts were RC; 38 after G1 ILD (81%), 9 after G2. Median (med) time to initial ILD was 145 days (d) after 1st dose (interquartile range [IQR] 78-205). Demographics for pts RC are shown in the table. Among 50 pts with G1 ILD, including pts not RC, 28/50 (56%) received steroids for a med of 36d (IQR 27-79). Radiographic improvement was seen at a med of 24d (IQR 19-63) for pts treated with steroids vs 82d (IQR 48-94) without (p<0.01); and a med of 35d (IQR 22-82) for pts RC vs 81d (IQR 68-105) for pts not RC (p=0.01). Among pts with G1 ILD, 38/50 (76%) were RC at a med of 42d (IQR 36-57) from last dose; 23/38 (61%) were dose reduced. After RC, pts remained on T-DXd for a med of 215d (IQR 60-334); 10/38 (26%) developed recurrent ILD (7-G1, 2-G2, 1-G3) at a med of 211d (IQR 47-273) from RC. No statistically significant differences were seen between ILD onset, time to RC, or demographics for pts with recurrent ILD vs not. Of the 9 pts RC after G2 ILD, T-DXd was continued for a med of 129d (IQR 49-171); 2/9 (22%) developed recurrent ILD (1-G2, 1-G3). No G5 toxicity was seen with RC. Conclusions: In this multi-center study, high RC rates were seen after G1 ILD with long duration of clinical benefit. Pts treated with steroids had faster radiographic ILD improvement, highlighting the importance of early steroid use. Among pts RC after G1 ILD, recurrent ILD rates were low, with the majority G1 and no G5 events. Notably, 9 pts with G2 ILD were RC, with a similar rate of recurrent ILD; this must be interpreted cautiously. Our large cohort data further supports the safety of T-DXd RC in diverse real-world settings. RC Pt Characteristics (n=47) n (%) or Median (IQR) Cancer typeBreastGIGyn 43 (91)3 (6)1 (2) Age (yrs) 57 (52-68) Prior # therapy lines in the advanced/metastatic setting 3 (1-5) Renal impairment (CrCl < 60 mL/min) 8 (17)

We sought to develop a survival model in chronic myelomonocytic leukemia (CMML) that is primarily based on clinical variables and examine additional impact from mutations and karyotype. 457 molecularly-annotated patients were considered. Multivariable analysis identified circulating Blasts ≥2% (1 point), Leukocytes ≥13 x 109/L (1 point), and severe (2 points) or moderate (1 point) Anemia as preferred risk variables in developing a clinical risk Stratification Tool for overall survival (OS), acronymized to "BLAST": low-risk (0 points; median 63 months); intermediate-risk (1 point; median 28 months; HR 2.2, 95% CI 1.6-3.0), and high-risk (2-4 points; median 13 months; 5.4, 4.1-7.3); the corresponding 3/5 year OS rates were 68%/53%, 43%/18%, and 12%/1%. BLAST model performance (AUC 0.77/0.85 at 3/5-years) was shown to be comparable to that of the molecular CMML-specific prognostic scoring system (CMML-mol; AUC 0.73/0.75) and the international prognostic scoring system-molecular (IPSS-M; AUC 0.73/0.74). Multivariable analysis of mutations and karyotype identified PHF6MUT and TET2MUT as being "favorable" and DNMT3AMUT, U2AF1MUT, BCORMUT, SETBP1MUT, ASXL1MUT, NRASMUT, PTPN11MUT, RUNX1MUT, TP53MUT, and adverse karyotype, "unfavorable". Molecular information was subsequently encoded in a combined clinical-molecular risk model (BLAST-mol; AUC 0.80/0.86 at 3/5-years) that included the aforementioned BLAST clinical risk variables and a 3-tiered molecular risk score. BLAST and BLAST-mol were subsequently validated by two separate external cohorts. Independent risk factors for blast transformation included DNMT3AMUT, ASXL1MUT, PHF6WT, leukocytes ≥13 x 109/L, and ≥2% circulating or ≥10% bone marrow blasts. The current study proposes an easy to implement, globally applicable, and molecularly adaptive risk model for CMML.

PURPOSE The combination of the Bruton tyrosine kinase inhibitor ibrutinib with bendamustine-rituximab for first-line treatment of mantle cell lymphoma (MCL) prolonged progression-free survival (PFS), but without improvement in overall survival (OS), likely because of toxicity. Acalabrutinib was shown to be efficacious and less toxic than ibrutinib in a head-to-head trial in chronic lymphocytic leukemia and therefore might lead to better outcomes in MCL. METHODS Patients 65 years and older with previously untreated MCL received acalabrutinib (100 mg twice daily) or placebo (until disease progression or unacceptable toxicity), plus six cycles of bendamustine (90 mg/m 2 once daily; days 1 and 2) and rituximab (375 mg/m 2 as a single dose; day 1) followed by rituximab maintenance in responding patients for 2 years. Crossover to acalabrutinib at disease progression was permitted. The primary end point was PFS per the independent review committee; overall response rate and OS were secondary end points. RESULTS In total, 598 patients were randomly assigned, with 299 in each arm. At a median follow-up of 49.8 months using the reverse Kaplan-Meier method, the median PFS was 66.4 months in the acalabrutinib arm and 49.6 months in the placebo arm (hazard ratio [HR], 0.73 [95% CI, 0.57 to 0.94]; P = .0160). Benefit was seen across all subgroups, including those with high-risk features. Overall response/complete response rates were 91.0%/66.6% and 88.0%/53.5% in the acalabrutinib and placebo arms, respectively. OS was not significantly different (HR, 0.86 [95% CI, 0.65 to 1.13]; P = .27). Grade 3 or greater adverse events were reported in 88.9% and 88.2% in the acalabrutinib and placebo arms, respectively. CONCLUSION The combination of acalabrutinib with bendamustine-rituximab significantly improved PFS. Clinical benefit of acalabrutinib with bendamustine-rituximab was achieved with manageable toxicity.

Abstract Background Morbidity from bacteremia may be exacerbated by inadequate initial antibiotic therapy. This concern however may lead to hesitance to de-escalate while awaiting pathogen identification and susceptibility results. We performed a randomized controlled trial evaluating the impact of a strategy for rapid pathogen and resistance detection directly from positive blood culture bottles (BCBs) using a rapid multiplex PCR (rmPCR) complemented with a rapid phenotypic screen (RPS) for extended-spectrum beta-lactamases and carbapenemases in a setting with moderate to high levels of antimicrobial resistance where routine direct-from-BCB disk diffusion susceptibility testing was in place Methods Patients with positive BCBs were randomized to 2 arms: 1) Standard BCB processing (including direct from BCB disk diffusion susceptibility testing - control, n=419) with antimicrobial stewardship (AS); or 2) rmPCR/RPS with AS (rmPCR/RPS, n=425). The primary outcome was time from positive blood culture result to prescription of an effective or optimal antibiotic regimen. An effective antibiotic regimen was defined as one active against the detected pathogen(s); an optimal antibiotic regimen was defined as an effective regimen which was also the most narrow spectrum and targeted one, as recommended by institutional guidelines. Mortality was analyzed as a secondary outcome. Results True bacteremia was identified in 658 patients (n=329 in both arms); 597 were monomicrobial (405 with aerobic or facultative anaerobic Gram negatives rods [AGNR]) and 61 polymicrobial (41 harboring AGNR). Time from BCB Gram stain to prescription of an effective antibiotic was 9.9 versus 3.2 hours in the control and rmPCR/RPS groups, respectively (p=0.58) and time from Gram stain to prescription of an optimal antibiotic regimen was 41.6 versus 29.4 hours in the two groups, respectively (p &lt; 0.001). 14-day mortality was 7.6% versus 4.3% (p = 0.069) and 30-day mortality was 11.2% versus 8.8% (p=0.30) in the two groups, respectively. Conclusion Compared to control methods which included direct-from-BCB disk diffusion susceptibility testing, an intervention comprising rmPCR/RPS with AS applied to positive blood cultures improved time to optimal therapy but not time to effective therapy or mortality. Disclosures Shawn Vasoo, MBBS, MRCP, D(ABP), D(ABIM) (Inf Dis), FRCPath, bioMerieux: In-kind, for this study|Rosco Diagnostica: In-kind, for this study Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria Paul Tambyah, MBBS (S'pore), Diplomate, American Board of Internal Medicine and Infectious Diseases, Moderna: Grant/Research Support|Sanofi-Pasteur: Grant/Research Support

ABSTRACT In our efforts to enhance sensitivity to PARP inhibitors, we identified clofarabine (CLF) as a potential therapy for drug-resistant ovarian cancer and nuclear trafficking of Cathepsin L (CTSL) as a treatment- responsive biomarker. Using PARP inhibitor-sensitive and -resistant OC cell lines, ex vivo cultures of patient-derived ovarian ascites (OVA), primary ovarian tumors, and xenografts (PDX), we found that CLF monotherapy induces nuclear CTSL (nCTSL) in CLF-responsive cells (CLF-r) and sensitizes them to PARP inhibitors olaparib and rucaparib. In CLF non-responsive cells (CLF-nr), a combination of CLF with olaparib is necessary for nCTSL trafficking and synergy. CLF+olaparib synergy was observed in 47% of CLF-r and 24% of CLF-nr OVA samples. Drug-induced nCTSL is crucial for DNA damage response, including cell cycle arrest and apoptosis. Knockdown of CTSL in both CLF-r and CLF-nr cells conferred resistance to the CLF+olaparib combination, emphasizing nCTSL’s role in the DNA damage response pathway (DDR). Mechanistically, CLF facilitates CTSL nuclear import via KPNB1 in CLF-r cells. In CLF-nr cells, both olaparib and CLF are needed to facilitate CTSL nuclear import. Additionally, CLF downregulates the nuclear export protein CRM1 (XPO1) in both cohorts. Interestingly, CLF does not downregulate CRM1 in a subset of OVAs (29%), and they were classified as CLF-resistant (CLF- Res). In these samples, inhibiting CRM1 with KPT8602 restored synergy between CLF and PARP inhibitors. In vivo, CLF-r and CLF-nr PDX models exhibited enhanced DDR, reduced tumor burden, and prolonged survival with the CLF+olaparib combination. These findings suggest the CLF+olaparib combination is a promising therapeutic strategy for drug-resistant OC by inducing DDR through CTSL nuclear localization.