Tumor Sensitivity Research Articles

Safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of KT-253, a targeted protein degrader of MDM2, in patients with relapsed/refractory (R/R) solid tumors, lymphoma, high grade myeloid malignancies and acute lymphoblastic leukemia (ALL).

3084 Background: The tumor suppressor p53 is mutated in approximately 50% of cancers. In those cancers with wild-type p53, its activity is controlled by mouse double minute 2 (MDM2), an E3 ligase that tags p53 for degradation. KT-253 is a novel, highly potent heterobifunctional MDM2 degrader that upregulates p53 activity and overcomes the p53-MDM2 feedback loop, resulting in >200-fold higher potency compared to MDM2 inhibitors. In preclinical PDX models of sensitive p53WT solid tumors, AML and ALL, KT-253 robustly activates p53, induces apoptosis, and results in tumor regressions with every 3-week dosing. The ability to rapidly induce an acute apoptotic response and dose intermittently may result in a therapeutic index that has eluded previous MDM2 targeting agents. Methods: The ongoing open-label Phase 1 study evaluates safety, PK, PD and preliminary efficacy of IV KT-253 administered on Day 1 of 21-day cycles. Patients (pts) with advanced (≥2 prior therapies) solid tumors (ST)/lymphomas (Arm A) and relapsed/refractory (R/R) high grade myeloid malignancies (AML, high/very-high risk myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms) and ALL (Arm B) are eligible. Blood samples are collected for KT-253 PK/PD analyses. Results: As of 26 January 2024, 18 pts have been treated, 13 in Arm A dose levels (A: DL) 1-4 and 5 in Arm B dose levels (B: DL) 1-2, with mean number of 3 and 2 doses, respectively. The most common solid tumor types were Merkel cell carcinoma ((MCC) n=3), adenoid cystic carcinoma ((ACC) n=2) and uveal melanoma (n=2). Arm B included 5 pts with R/R AML. The median age was 61 years (yrs) (range 42, 81) in Arm A and 66 yrs (range 57, 70) in Arm B. The most common adverse events (AEs) in > 20% of pts included nausea, fatigue, headache, and vomiting. There was 1 DLT of AEs leading to discontinuation that included Grade (G) 2 nausea and fatigue in A: DL4. There were no neutropenia or thrombocytopenia AEs in either arm. KT-253 related SAEs included G3 hypotension in a pt with decreased oral intake (A: DL1). Overall best response: 1 CR (B: DL2, AML); 2 PRs (A: DL1, MCC; B: DL1, AML); 3 SD (A: DL1, fibromyxoid sarcoma; DL2, ACC; DL3, renal cell cancer). PD data from A: DL1-4 and B: DL1-2 demonstrated rapid upregulation of plasma GDF-15 protein and upregulation of CDKN1A and PHLDA3 mRNA levels in blood. KT-253 demonstrated dose-dependent increase in plasma exposure with levels approximating efficacious doses. Conclusions: KT-253 results in potent upregulation of p53-dependent biomarkers and has demonstrated early signs of anti-tumor activity including objective responses in MCC and AML at doses that are well tolerated. Dose escalation is ongoing at DL4 in Arm A and at DL2 in Arm B and analyses from additional pts will be presented at the meeting. Clinical trial information: NCT05775406 .

Development and validation of AI-assisted transcriptomic signatures to personalize adjuvant chemotherapy in patients with resectable pancreatic ductal adenocarcinoma.

4015 Background: Adjuvant chemotherapies for PDAC include modified FOLFIRINOX (mFFX) or gemcitabine-based regimen in fit patients and gemcitabine or 5FU single agents in other patients. While more effective, mFFX is associated with a greater toxicity than other options. Moreover, therapeutic decisions still rely mainly on the patient's performance status rather than tailored to tumor-based criteria. Our study aims to personalize treatments by developing transcriptomic signatures specific to commonly used drugs for pancreatic cancer. Methods: We analyzed the response to drugs (5-fluorouracil, oxaliplatin, and irinotecan) in three types of preclinical models (primary cell cultures, tumoroids, and patient-derived xenografts in immunodeficient mice). We then associated the detected sensitivities to drugs with transcriptomic data from each model. We also incorporated the previously developed gemcitabine signature. Finally, we used a machine learning method, the "Least Absolute Shrinkage and Selection Operator-random forest," to improve the signatures, integrating the tumor microenvironment master regulators. The learning cohort were GemPred for gemcitabine (1) and COMPASS (2) for mFFX. The resulting transcriptomic predictive tool was called Pancreas-View. We validated these signatures in the PRODIGE-24/CCTG PA6 trial cohort comprising 343 patients (3). Results: The signatures may allow to identify responsive patients to specific drugs and showed a significant improvement in their cancer-specific survival (CSS) and disease-free survival (DFS) when they received a matched therapy (mFFX or gemcitabine). Additionally, a positive association was observed between the number of drugs for which tumors predict to be sensitive and patient’s survival when appropriately treated. Patients who received “appropriate” drugs (n = 164; 47.8%) displayed a longer DFS : 50.1 months (stratified HR: 0.31; 95% CI, 0.21-0.44; p < 0.001) in the mFFX arm, and 33.7 months (stratified HR: 0.40; 95% CI, 0.17-0.59; p < 0.001) in the gemcitabine arm, respectively. Conversely, patients that received a treatment not matched with the signature prediction (n = 86; 25.1%) and those predicted to be resistant to all drugs (n = 93; 27.1%) had the poorest DFS results (10.6 and 10.8 months, respectively). Conclusions: By integrating preclinical models and machine learning, we developed a comprehensive predictive tool based on the transcriptome that may help to identify tumors sensitivity to mFFX components and gemcitabine. Crucially, these transcriptomic signatures can also lead to reduce toxicity by avoiding the unnecessary administration of drugs predicted as ineffective for a given tumor. Nicolle R, et al. Ann Oncol 2021;32:250-260. Aung K, et al Clin Cancer Res 2018;24:1344–1354. Conroy T, et al. N Engl J Med 2018; 379:2395-2406.

Little ones can do big things: Small molecule inhibitors target PTPN2/PTPN1 for tumor immunotherapy.

AC484 was developed by designing compounds based on the PTPN2 protein structure. AC484 enhances antitumor immunity through multiple mechanisms: increasing tumor sensitivity to IFN-γ, improving T-cell functions, stimulating tumor microenvironment inflammation, expanding TCR diversity, and preventing T-cell exhaustion. Interestingly, the efficacy of AC484 was also mediated by CD8+ and NK cells.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

Long Non-Coding RNAs in Drug Resistance of Gastric Cancer: Complex Mechanisms and Potential Clinical Applications.

Gastric cancer (GC) ranks as the third most prevalent malignancy and a leading cause of cancer-related mortality worldwide. However, the majority of patients with GC are diagnosed at an advanced stage, highlighting the urgent need for effective perioperative and postoperative chemotherapy to prevent relapse and metastasis. The current treatment strategies have limited overall efficacy because of intrinsic or acquired drug resistance. Recent evidence suggests that dysregulated long non-coding RNAs (lncRNAs) play a significant role in mediating drug resistance in GC. Therefore, there is an imperative to explore novel molecular mechanisms underlying drug resistance in order to overcome this challenging issue. With advancements in deep transcriptome sequencing technology, lncRNAs-once considered transcriptional noise-have garnered widespread attention as potential regulators of carcinogenesis, including tumor cell proliferation, metastasis, and sensitivity to chemo- or radiotherapy through multiple regulatory mechanisms. In light of these findings, we aim to review the mechanisms by which lncRNAs contribute to drug therapy resistance in GC with the goal of providing new insights and breakthroughs toward overcoming this formidable obstacle.

Tumor biomarkers for diagnosis, prognosis and targeted therapy.

Tumor biomarkers, the substances which are produced by tumors or the body's responses to tumors during tumorigenesis and progression, have been demonstrated to possess critical and encouraging value in screening and early diagnosis, prognosis prediction, recurrence detection, and therapeutic efficacy monitoring of cancers. Over the past decades, continuous progress has been made in exploring and discovering novel, sensitive, specific, and accurate tumor biomarkers, which has significantly promoted personalized medicine and improved the outcomes of cancer patients, especially advances in molecular biology technologies developed for the detection of tumor biomarkers. Herein, we summarize the discovery and development of tumor biomarkers, including the history of tumor biomarkers, the conventional and innovative technologies used for biomarker discovery and detection, the classification of tumor biomarkers based on tissue origins, and the application of tumor biomarkers in clinical cancer management. In particular, we highlight the recent advancements in biomarker-based anticancer-targeted therapies which are emerging as breakthroughs and promising cancer therapeutic strategies. We also discuss limitations and challenges that need to be addressed and provide insights and perspectives to turn challenges into opportunities in this field. Collectively, the discovery and application of multiple tumor biomarkers emphasized in this review may provide guidance on improved precision medicine, broaden horizons in future research directions, and expedite the clinical classification of cancer patients according to their molecular biomarkers rather than organs of origin.

Novel imidazolone derivatives as potential dual inhibitors of checkpoint kinases 1 and 2: Design, synthesis, cytotoxicity evaluation, and mechanistic insights

Applying various drug design strategies including ring variation, substituents variation, and ring fusion, two series of 2-(alkylthio)-5-(arylidene/heteroarylidene)imidazolones and imidazo[1,2-a]thieno[2,3-d]pyrimidines were designed and prepared as dual potential Chk1 and Chk2 inhibitors. The newly synthesized hybrids were screened in NCI 60 cell line panel where the most active derivatives 4b, d-f, and 6a were further estimated for their five dose antiproliferative activity against the most sensitive tumor cells including breast MCF-7 and MDA-MB-468 and non-small cell lung cancer EKVX as well as normal WI-38 cell. Noticeably, increasing the carbon chain attached to thiol moiety at C-2 of imidazolone scaffold elevated the cytotoxic activity. Hence, compounds 4e and 4f, containing S-butyl fragment, exhibited the most antiproliferative activity against the tested cells where 4f showed extremely potent selectivity toward them. As well, compound 6a, containing imidazothienopyrimidine core, exerted significant cytotoxic activity and selectivity toward the examined cells. The mechanistic investigation of the most active cytotoxic analogs was achieved through the evaluation of their inhibitory activity against Chk1 and Chk2. Results revealed that 4f displayed potent dual inhibition of both Chk1 and Chk2 with IC50 equal 0.137 and 0.25 μM, respectively. It also promoted its antiproliferative and Chk suppression activity via EKVX cell cycle arrest at S phase through stimulating the apoptotic approach. The apoptosis induction was also emphasized by elevating the expression of Caspase-3 and Bax, that are accompanied by Bcl-2 diminution. The in silico molecular docking and ADMET profiles of the most active analogs have been carried out to evaluate their potential as significant anticancer drug candidates.

A head-to-head comparison of [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI PET/CT in patients with nasopharyngeal carcinoma: a single-center, prospective study.

We aimed to compare the staging efficiency of [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI PET/CT in nasopharyngeal carcinoma (NPC) patients. Thirty-nine patients with pathologically confirmed NPC were enrolled in this prospective study. Each patient underwent paired [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI PET/CT on 2 successive days. The accuracy of two PET/CT for assessing T, N, and M stages was compared by using head-and-neck MRI, histopathologic diagnosis and follow-up results as reference standards. The radiotracer uptake derived from two PETs was also compared. For treatment-naïve patients, [68Ga]Ga-DOTATATE PET/CT showed identical sensitivity for the primary tumours but clearer tumor delineation induced by higher tumour-to-background (TBR) ratio (19.1 ± 8.7 vs. 12.4 ± 7.7, P = 0.003), compared with [68Ga]Ga-FAPI PET/CT. Regarding cervical lymph node (CLN) metastases, [68Ga]Ga-DOTATATE PET had significantly better sensitivity and accuracy based on neck sides (98% vs. 82%, P < 0.001; 99% vs. 88% P = 0.008), neck levels (98% vs. 78%, 99% vs. 97%; both P < 0.001) and individual nodes (89% vs. 56%, 91% vs. 76%; both P < 0.001), and higher TBR (8.1 ± 4.1 vs. 6.3 ± 3.7, P < 0.001). Additionally, [68Ga]Ga-DOTATATE PET/CT revealed higher sensitivity and accuracy for distant metastases (96% vs. 53%, 95% vs. 52%; both P < 0.001), particularly in bone metastases (99% vs. 49%, 97% vs. 49%; both P < 0.001). For post-treatment patients, [68Ga]Ga-DOTATATE PET/CT identified one more true-negative case than [68Ga]Ga-FAPI PET/CT. [68Ga]Ga-DOTATATE PET/CT performed better than [68Ga]Ga-FAPI PET/CT in visualizing the primary tumours, detecting the metastatic lesions and identifying the local recurrence, suggesting [68Ga]Ga-DOTATATE PET/CT may be superior to [68Ga]Ga-FAPI PET/CT for NPC staging.

Long-term combined blockade of CXCR4 and PD-L1 with in vivo reassembly for intensive tumor interference

Negative immunoregulatory signal (PD-L1, CXCR4, et al.) and weak immunogenicity elicited immune system failing to detect and destroy cancerous cells. CXCR4 blockade promoted T cell tumor infiltration and increased tumor sensitivity to anti-PD-L1 therapy. Here, pH-responsive reassembled nanomaterials were constructed with anti-PD-L1 peptide and CXCR4 antagonists grafting (APAB), synergized with photothermal therapy for melanoma and breast tumor interference. The self-assembled APAB nanoparticles accumulated in the tumor and rapidly transformed into nanofibers in response to the acidic tumor microenvironment, leading to the exposure of grafted therapeutic agents. APAB enabling to reassemble around tumor cells and remained stable for over 96 h due to the aggregation induced retention (AIR) effect, led to long-term efficiently combined PD-L1 and CXCR4 blockade. Photothermal efficiency (ICG) induced immunogenic cell death (ICD) of tumor cells so as to effectively improve the immunogenicity. The combined therapy (ICG@APAB) could effectively inhibit the growth of primary tumor (∼83.52%) and distant tumor (∼76.24%) in melanoma-bearing mice, and significantly (p < 0.05) prolong the survival time over 42 days. The inhibition assay on tumor metastasis in 4 T1 model mice exhibited ICG@APAB almostly suppressed the occurrence of lung metastases and the expression levels of CD31, MMP-9 and VEGF in tumor decreased by 82.26%, 90.45% and 41.54%, respectively. The in vivo reassembly strategy will offer novel perspectives benefical future immunotherapies and push development of combined therapeutics into clinical settings.

Abstract PO1-15-06: Serum thymidine kinase activity as a “real-time” clinical biomarker of tumor response to CDK4/6 inhibition in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer

Abstract Background: CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) are standard first-line (1L) treatment for hormone receptor positive (HR+), HER2-negative (HER2-) metastatic breast cancer (MBC). Despite general improvements in long-term outcome, not all patients benefit equally from these drugs. The average duration of 1L CDK4/6i therapy is ~2 years. However, 10-15% of patients will progress within 6 months of therapy initiation, while others will derive durable clinical benefit of years. Differences in treatment efficacy cannot be easily explained by clinical-pathological features. No predictive biomarkers are currently available to determine tumor sensitivity to CDK4/6i. There is an unmet need for a complementary diagnostic to identify patients that will or will not respond to these treatments in order to better inform therapy management. Thymidine kinase (TK) is an E2F-regulated enzyme that plays a key role in DNA replication during cell division. The expression and activity of TK is strongly linked to the cell cycle. TK is first expressed in late G1, peaks during S-phase, and then is degraded during mitosis. Because of the restricted expression of TK (only during S phase of the cell cycle) combined with the mechanism of action of CDK4/6i, the measurement of TK activity (TKa) may serve as an ideal pharmacodynamic biomarker to assess a tumor’s response to CDK4/6i-based therapy. DiviTum® TKa is an FDA cleared assay that can quantitate TKa from human serum (sTKa) and is intended to be used as a disease progression monitoring tool for patients with HR+, HER2- MBC. TK IMPACT is a prospective, single arm trial designed to assess the impact of incorporation of DiviTum® TKa on the physician’s decision regarding subsequent timing of routine disease monitoring modalities in patients with advanced HR+, HER2- MBC receiving 1L ET plus CDK 4/6i (NCT04968964). As a part of this ongoing study, DiviTum® TKa values are serially assessed relative to imaging findings and other circulating tumor biomarkers, such as CA15-3 and ctDNA. The primary endpoint is any physician-reported intended change in imaging testing interval post TKa by study cohort, within the first 48-week period of study participation, assessments for which are ongoing. Here we report real-world data and examples of the clinical utility of sTKa as a companion diagnostic from patients treated at Washington University Siteman Cancer Center, St. Louis, MO. Methods: Patients with HR+, HER2- MBC receiving 1L ET + any FDA-approved CDK 4/6i (palbociclib, ribociclib, or abemaciclib) had blood serum samples collected at baseline, weeks 2, 4, 6, 8, every 4 weeks up to week 24, and then every 12 weeks thereafter until disease progression. Samples were analyzed for TKa levels in real-time using the DiviTum® TKa assay. Optional repeat TKa within 2-4 wks (+/- 3 days) is permitted in case of rising TKa. Other standard monitoring assessments were carried out according to institutional guidelines and/or treating clinician discretion. Results: Pre-treatment and early on-therapy TKa levels predicted response to CDK4/6i therapy more accurately than a patient’s clinical characteristics. Rising TKa levels also identified patients with clinically occult disease progression ahead of imaging, and in some cases where radiographic interpretation and serum tumor markers such as CA15-3 were ambiguous. Measurement of TKa levels following CDK4/6i dose reduction was able to confirm continued optimal therapy response at the lower dose. A rise in TKa levels aligned with the re-emergence of ESR1 mutation as identified by ctDNA in one patient and may herald molecular progression. Conclusion: sTKa was able to predict tumor sensitivity and resistance (both intrinsic and acquired) to CDK4/6i + ET therapy in patients with HR+, HER2- MBC. We believe these findings help support the role of sTKa as a non-invasive pharmacodynamic biomarker that can assess in real-time a tumor’s response to CDK4/6i based therapy. Citation Format: Nusayba Bagegni, Amy Williams, Isabella Grigsby, Mattias Bergqvist, Katherine Clifton, Cynthia Ma. Serum thymidine kinase activity as a “real-time” clinical biomarker of tumor response to CDK4/6 inhibition in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-06.

Abstract PO2-14-11: Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment

Abstract NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader (SERD), together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ PDX models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pull-down. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An immunohistochemistry (IHC) assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Citation Format: Beom-Jun Kim, Ze-Yi Zheng, Jonathan Lei, Matthew Holt, Anran Chen, Jianheng Peng, Diana Fandino, Purba Singh, Hilda Kennedy, Yongchao Dou, M Rosario Chica-Parrado, Emmanuel Bikorimana, Dan Ye, Yunguan Wang, Ariella Hanker, Nada Mohamed, Susan Hilsenbeck, Bora Lim, Jaya Ruth Asirvatham, Arun Sreekumar, Bing Zhang, George Miles, Meenakshi Anurag, Matthew Ellis, Eric Chang. Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-11.

Abstract RF01-05: Functional assessment of RAD51 foci and replication fork dynamics in PARPi resistant BRCA1/2 mutated breast cancer

Abstract Background: Although platinum salts (Pt) or Poly (ADP-Ribose) Polymerase inhibitors (PARPi) are effective in treating homologous recombination defective (HRD) breast cancer, resistance often emerges, especially in advanced disease. Predicting response and relapse is complex, even in patients with germline BRCA1/2 mutations (gBRCA1/2m). Clinically approved HRD detection methods are limited to identification of pathogenic mutations in HR genes or mutational signatures in the genome of tumors caused by HRD. In PDX models derived from HRD breast cancer, the restoration of nuclear RAD51 foci formation, a key feature of functional HR, can predict resistance to HRD-targeted treatment.1 In addition, the restoration of replication fork stability, despite PARPi or induction of Pt adducts that induce replication fork arrest and collapse, confers resistance in pre-clinical models2; to date this resistance mechanism has not been clinically validated. Here we analyse RAD51 foci in FFPE samples and DNA replication fork dynamics, using DNA fibre combing assays, in patient derived organoids (PDO) relating these to clinical response to illustrate how such assays might predict clinical HRD-targeted therapy resistance in metastatic breast cancer (MBC). Patients and Methods: We used immunofluorescent detection of RAD51 foci as a marker for HR proficiency (HRP) in tumors from 29 patients with gBRCA1/2m with MBC treated with HRD-targeted treatment (n=6 PARPi, n=2 Pt and n=21 both agents in sequence). All patients developed resistance that was either de novo or acquired. RAD51 and BRCA1 foci were scored in a minimum of 50 geminin (a marker of S/G2 phase) positive tumor cells; cells with ≥5 foci were classified as positive, and tumours where ≤10% or &amp;gt;10% of cells were positive were considered HRD or HRP, respectively. DNA replication fork dynamics were assessed in with or without PARPi using thymidine analogue labelling. DNA fibre analysis was performed in PDOs developed from fresh tumor sampling of HRD-targeted treatment sensitive or resistant tumors, to determine the relationship between replication fork dynamics, stability and PARPi sensitivity. Results: RAD51 analysis was performed on 9 treatment naïve samples (n=9 patients), 8 samples obtained after HRD-targeted treatment resistance (n=8 patients) and 27 samples obtained pre and post HRD-targeted treatment resistance (n=12 patients). Functional HRD by RAD51 in treatment naive samples was seen in 100% (n=17) of patients with acquired resistance and 66% of patients with de novo resistance. All patients, whether with de novo or acquired resistance, exhibited high RAD51 scores in post-resistance tumour samples, suggesting restoration of HR function is the dominant mechanism of PARPi resistance. As such, RAD51 analysis shows potential as a biomarker of clinical PARPi resistance. Replication fork stability fibre was analysed after exposure to potent PARP1 trapping inhibitors or an ATRi control in 3 PDOs (2 gBRCA1m, 1 BRCAwt). As controls we used an isogenic 2D cell line with and without a CRISPR engineered BRCA1 reversion mutation (SUM149 parental BRCA1m and SUM149BS*1 revertant) or with a PARP1 mutation (SUM149 TR2 clone) that prevents PARP1 being trapped on DNA. These experiments indicate that replication fork dynamics can be assessed in “patient derived” models of breast cancer and that PARPi sensitivity was associated with PARPi induced replication fork instability (median ratio IdU:CldU in resistant lines: 0.98, sensitive lines: 0.60). Conclusion: We show that HRP restoration and RAD51 foci in advanced BRCA1/2m breast cancers is the dominant form resistance to HRD-targeted treatment. We also demonstrate for the first time that analysis of DNA replication fork dynamics can be carried out in breast cancer PDOs and could be further explored as a functional predictive biomarker of PARPi resistance. 1.Pellegrino et al, 2022 (PMID: 35425960) 2.Chaudhuri et al, 2016 (PMID: 27443740) Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Thanussuyah Alaguthurai, Alba Llop-Guevara, Jennifer Trendell, Olga Rodriguez, Marta Guzman, Daniel Weekes, Christelle deRenty, Eleanor Knight, Rebecca Marlow, Christopher Starling, Ruifang Liu, Nivedita Ravindran, Ioannis Roxanis, Natalia Lukashchuk, Violeta Serra, Stephen Pettitt, Christopher Lord, Andrew NJ Tutt. Functional assessment of RAD51 foci and replication fork dynamics in PARPi resistant BRCA1/2 mutated breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-05.

Abstract PO3-07-10: Advanced CD8 ImmunoPET imaging predicts radiation response in primary TNBC

Abstract Background: Tumor immunology and immunosuppression has been observed to drive changes in intratumoral response to cytotoxic and immunotherapy in triple negative breast cancer (TNBC). While radiation therapy is standard of care for TNBC, there is a lack of research into the relationship between long term treatment response and CD8 immune infiltration in the context of a radiation resistant model. Positron emission tomography (PET) imaging allows for noninvasive monitoring of the tumor microenvironment that can precede changes in tumor volume, including approaches that allow for immune imaging of CD8 T-cell trafficking. Noninvasive PET imaging of radiation response has the potential to identify windows of enhanced response to secondary therapy and can guide interventional therapy. The goal of this study is to understand how radiation can prime the tumor microenvironment to be combined with other therapeutics in TNBC through changes in CD8 immune infiltration with [89Zr]-CD8 ImmunoPET imaging. Methods: Syngeneic parental 4T1 (radiation sensitive) and a developed radiation-resistant 4T1 sub-cell line were orthotopically injected into BALB/c mice (N = 5 control, N = 9-10 radiation treated for each model). After reaching a tumor volume of ~100 mm3, mice began treatment with 2 Gy fractionated radiation daily from day 0-5. On day 6, mice were injected with ~50 µCi of [89Zr]-CD8 minibody (IAB42) and imaged 24 hours post injection. The mean standardized uptake value (SUV) was quantified and normalized to heart SUV to extract out a tumor:heart SUVratio that describe the CD8 infiltration within tumors. Following imaging, tumors were either excised immediately for immunofluorescence against CD8 or monitored for longitudinal changes in tumor volume. A non-parametric T-test was used to assess for significance between groups. Results: In radiation sensitive 4T1 tumors, [89Zr]-CD8 ImmunoPET imaging indicated a 23% increase in CD8 immune infiltration, in radiation-treated tumors compared to control tumors on day 7 (p = 0.02). These immune alterations occurred prior to any changes in tumor volume (p = 0.63). Longitudinally, radiation treated tumors exhibited significant changes in tumor volume beginning on day 20 (p = 0.02), which is sustained until study endpoint on day 41 (p &amp;lt; 0.01). In radiation resistant 4T1 tumors, no significant change was observed in short term CD8 immune infiltration (p = 0.43) or in longitudinal tumor volume (p = 0.31) in response to radiation therapy, when compared to untreated tumors. Conclusions: [89Zr]-CD8 ImmunoPET imaging reveals significant increases in CD8 immune infiltration that is predictive of eventual response to radiation therapy in radiation sensitive models of TNBC. [89Zr]-CD8 PET imaging of radiation therapy response has the potential to increase the efficacy of precision medicine and prime the tumor microenvironment for secondary therapeutics, thereby improving tumor kill and reducing patient toxicity. Citation Format: Patrick Song, Shannon Lynch, Chloe DeMellier, Alessandro Mascioni, Fang Jia, Anna Sorace. Advanced CD8 ImmunoPET imaging predicts radiation response in primary TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-07-10.

Abstract PO1-07-12: Inverse modeling with surface temperature accurately detects the presence of breast cancer

Abstract Breast cancer affects over 250,000 women in the United States every year. Patient outcomes including overall survival significantly decline with large sized tumors and nodal involvement on initial presentation. The introduction of mammogram screening has been instrumental in the early detection of cancer. However, it has lower sensitivity in dense breast tissue and limited specificity which leads to unnecessary additional invasive testing. Several methods such as magnetic resonance imaging (MRI), computed tomography (CT) and ultrasound imaging are used as adjunctive methods. However, there is still a need for cost-effective techniques without additional radiation exposure for breast cancer screening for use with mammography. We propose an adjunctive screening method using surface temperature measurements and inverse breast modeling for early detection of breath cancer. Methods: We enrolled patients who presented with mammogram detected breast tumors that were confirmed to be cancerous lesions by biopsy. We used prone position steady state infrared imaging of bilateral breasts to measure surface temperatures and multi-view MRI images to create patient specific 3D breast models. The inverse technique applies Levenberg-Marquard algorithms (LMA) and utilizes commercial software for thermal transport modeling. The inverse technique predicts a heat generation map normalized for baseline surface temperatures to localize the presence of an underlying tumor. Results: A total of 25 breast tumors (diameter range from 5 to 27 mm) from 24 patients were included in the analysis with a median patient age of 67 years. One patient had bilateral breast tumors. Mammogram data showed inclusion of mixed breast tissue composition including dense and extremely dense breast tissue. LMA accurately detected the tumor in all 25 patients with maximum absolute errors of 7 mm in location of the tumor in the breast and 1.78 mm in diameter. The accuracy of detection was not affected by tumor histology which included invasive ductal and lobular carcinomas, ductal and lobular carcinoma in-situ and atypical ductal hyperplasia. No heat sensitive tumors were detected in the 23 contralateral breasts which acted as internal negative controls. Conclusion: Infrared temperature profiles and inverse modeling successfully detected malignant breast tumors with no missed tumors or false positive results and could be used as adjunctive screening along with mammography, especially in patients with dense breast tissue. Citation Format: Nithya Sritharan, Carlos Gutierrez, Isaac Perez-Raya, Jose-Luis Gonzalez-Hernandez, Alyssa Owens, Donnette Dabydeen, Lori Medeiros, Satish Kandlikar, Pradyumna Phatak. Inverse modeling with surface temperature accurately detects the presence of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-07-12.

Abstract PO1-07-02: Sensitive tumor detection, accurate quantification, and ER subtyping using low-pass methylome of liquid biopsy samples from patients (pts) with metastatic breast cancer (mBC)

Abstract Background The presence and amount of blood circulating tumor DNA (Tumor Content, TC) is emerging as a clinically relevant factor in many cancers, including BC. In the metastatic setting, a commonly used tumor tissue agnostic approach involves analyzing Copy Number Alterations (CNA) from low-pass whole-genome sequencing (lpWGS) by ichorCNA. However, with a lower limit of detection of 3%, it is unsuitable for low TC applications, such as monitoring disease in response to treatment. Here we report on the use of low-pass whole genome bisulfite sequencing (lpWGBS) coupled with a novel tool, METER (METhylation analyzER), to overcome current limitations of lpWGS, enabling sensitive TC detection, accurate quantification, and ER subtyping from mBC liquid biopsies. Methods METER is a computational tool to analyse TC exploiting BC-specific Differentially Methylated Sites (DMS) and Regions (DMR) in lpWGBS data. To define BC DMSs and DMRs, Rocker-meth was applied to the WGBS profiles of 30 BC tissue (BASIS dataset) and 23 healthy donor cell free DNA (cfDNA) (Fox-Fisher 2021). METER consists of three modules: 1) METER-quant, a DMS-based quantifier of TC; 2) METER-detect, a DMR-based z-score method to classify samples as TC+ (METER+) or TC- (METER-); 3) METER-subtype, to infer ER status via Robust Partial Correlation applied to ER+ vs. ER- BC DMR. We generated lpWGBS data (coverage 0.5-1X) of 135 cfDNA samples from 58 pts (45 ER+, 13 ER-, 55 (95%) up to 3 previous lines of treatment; before starting treatment (T0, n=58), after the first cycle (T1, n=25), and at progression (T2, n=22)) and 30 from healthy individuals as controls. ichorCNA using sensitive parameters (allowing normal fraction up to 0.99) was used to estimate reference TC. A leave-one-out strategy applied to the control samples was used to estimate the false discovery rate (FDR) of METER-detect classification. METER+/- pts were tested for association with OS and PFS using the log-rank test and compared with ichorCNA detection (TC by ichorCNA above or below 3%, ichorCNA+/-). Results TC of 105 cfDNA BC samples by METER-quant was concordant with estimates from the state-of-the-art tool ichorCNA (R &amp;gt;0.90, p&amp;lt; 1e-10). In 22 pts with complete longitudinal data, METER-quant median TC of 0.05 at T0 (IQR 0.02-0.12), 0.02 at T1 (0.01-0.07), and 0.06 at T2 (0.03-0.11) were obtained, with significant difference observed for T0 vs T1 and T2 vs T1 (paired Wilcoxon p&amp;lt; 0.05). Across all time points, METER-detect classified 43% of the n=42 ichorCNA- samples as METER+. The reliability and prognostic performance of METER and ichorCNA detection classification were then compared using clinical outcome data. Based on T0 data, METER+ pts had significantly worse OS than METER- (HR=4.2 CI=2.0-8.9, p&amp;lt; 0.001). This effect was stronger than using ichorCNA (HR=2.3 CI=1.3-4.0, p=0.006). Worse PFS at T0 was observed for METER+ compared with METER- pts (HR=3.7 CI=1.8-7.9, p&amp;lt; 0.001), while no significant association was observed for ichorCNA+ pts (HR=1.4 CI=0.8-2.5, p=0.2). Of note, ichorCNA-/METER+ pts (N=11) at T0 had worse PFS compared with ichorCNA-/METER- (N=11) (HR=3.7 CI 1.4-9.7, p=0.005). Considering the 73 METER+ and 53 samples with METER-quant &amp;gt;5%, METER-subtype showed an accuracy of 0.84 (CI=0.73-0.92) and 0.94 (CI=0.83-0.99) respectively in classifying ER status based on IHC. Discussion lpWGBS enables tumor tissue agnostic analysis and concurrent investigation of complementary molecular features, including CNA and DNA methylation patterns. In a small and heterogeneous cohort, METER showed comparable performance to state-of-the-art tools in terms of TC quantification (ichorCNA) and ER subtyping (IHC), while offering enhanced sensitivity and ensuring a low FDR. METER showed promising prognostic stratification capabilities, particularly for patients with low TC. Validation in an extended prospective cohort is currently ongoing. Citation Format: Marta Paoli, Agostina Nardone, Francesca Galardi, Chiara Biagioni, Dario Romagnoli, Francesca De Luca, Gian Marco Franceschini, Ilenia Migliaccio, Marta Pestrin, Giuseppina Sanna, Emanuela Risi, Luca Livraghi, Erica Moretti, Laura Biganzoli, Luca Malorni, Francesca Demichelis, Matteo Benelli. Sensitive tumor detection, accurate quantification, and ER subtyping using low-pass methylome of liquid biopsy samples from patients (pts) with metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-07-02.

Integration of Computational Pipeline to Streamline Efficacious Drug Nomination and Biomarker Discovery in Glioblastoma.

Glioblastoma multiforme (GBM) is the deadliest, most heterogeneous, and most common brain cancer in adults. Not only is there an urgent need to identify efficacious therapeutics, but there is also a great need to pair these therapeutics with biomarkers that can help tailor treatment to the right patient populations. We built patient drug response models by integrating patient tumor transcriptome data with high-throughput cell line drug screening data as well as Bayesian networks to infer relationships between patient gene expression and drug response. Through these discovery pipelines, we identified agents of interest for GBM to be effective across five independent patient cohorts and in a mouse avatar model: among them are a number of MEK inhibitors (MEKis). We also predicted phosphoglycerate dehydrogenase enzyme (PHGDH) gene expression levels to be causally associated with MEKi efficacy, where knockdown of this gene increased tumor sensitivity to MEKi and overexpression led to MEKi resistance. Overall, our work demonstrated the power of integrating computational approaches. In doing so, we quickly nominated several drugs with varying known mechanisms of action that can efficaciously target GBM. By simultaneously identifying biomarkers with these drugs, we also provide tools to select the right patient populations for subsequent evaluation.

Improving tumor sensitivity by the introduction of an ester chain to triaryl derivatives targeting PD-1/PD-L1

PD-1/PD-L1 pathway blockade is a promising immunotherapy for the treatment of cancer. In this manuscript, a series of triaryl compounds containing ester chains were designed and synthesized based on the pharmacophore studies of the lead BMS-1. After several SAR iterations, 22 showed the best biochemical activity binding to hPD-L1 with an IC50 of 1.21 nM in HTRF assay, and a KD value of 5.068 nM in SPR analysis. Cell-based experiments showed that 22 effectively promoted A549 cell death by restoring T-cell immune function. 22 showed significant in vivo antitumor activity in a 4T1 mouse model without obvious toxicity, with a TGI rate of 67.8 % (20 mg/kg, ip). Immunohistochemistry data indicated that 22 activates the immune activity in tumors. These results suggest that 22 is a promising compound for further development of PD-1/PD-L1 inhibitor for cancer therapy.

TFAP2A Upregulates SKA3 to Promote Glycolysis and Reduce the Sensitivity of Lung Adenocarcinoma Cells to Cisplatin

Introduction: Studies have shown that glycolysis metabolism affects the resistance or sensitivity of tumors to chemotherapy drugs. Emerging from recent research, a paradigm-shifting revelation has unfolded, elucidating the oncogenic nature of SKA3 within the context of lung adenocarcinoma (LUAD). Consequently, this work was designed to delve into the effects of SKA3 on glycolysis and cisplatin (CDDP) resistance in LUAD cells and to find new possibilities for individualized treatment of LUAD. Methods: LUAD mRNA expression data from the TCGA database were procured to scrutinize the differential expression patterns of SKA3 in both tumor and normal tissues. GSEA and Pearson correlation analyses were employed to elucidate the impact of SKA3 on signaling pathways within the context of LUAD. In order to discern the upstream regulatory mechanisms, the ChEA and JASPAR databases were utilized to predict the transcription factors and binding sites associated with SKA3. qRT-PCR and Western blot were implemented to assay the mRNA and protein expression levels of SKA3 and TFAP2A. Chromatin immunoprecipitation and dual-luciferase assays were performed to solidify the binding relationship between the two. Extracellular acidification rate, glucose consumption, lactate production, and glycolysis-related proteins (HK2, GLUT1, and LDHA) were used to evaluate the level of glycolysis. Cell viability under CDDP treatment was determined utilizing the CCK-8, allowing for the calculation of IC50. The expression levels of SKA3 and TFAP2A proteins were detected by immunohistochemistry (IHC). Results: SKA3 exhibited upregulation in LUAD tissues and cell lines, establishing a direct linkage with glycolysis pathway. Overexpression of SKA3 fostered glycolysis in LUAD, resulting in reduced sensitivity toward CDDP treatment. The upstream transcription factor of SKA3, TFAP2A, was also upregulated in LUAD and could promote SKA3 transcription. Overexpression of TFAP2A also fostered the glycolysis of LUAD. Rescue assays showed that TFAP2A promoted glycolysis in LUAD cells by activating SKA3, reducing the sensitivity of LUAD cells to CDDP. The IHC analysis revealed a positive correlation between high expression of SKA3 and TFAP2A and CDDP resistance. Conclusion: In summary, TFAP2A can transcriptionally activate SKA3, promote glycolysis in LUAD, and protect LUAD cells from CDDP treatment, indicating that targeting the TFAP2A/SKA3 axis may become a plausible and pragmatic therapeutic strategy for the clinical governance of LUAD.

The heterogeneous sensitivity of pediatric brain tumors to different oncolytic viruses is predicted by unique gene expression profiles

Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal, however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124 and the non-virulent Newcastle Disease Virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.

An enzymatically activated self-powered and self-recycled strategy for in situ tumor cell-specific molecular imaging

Specific imaging of tumor cells with high sensitivity is urgently needed for evaluating the risk of tumor metastasis and progression. However, traditional tumor cell imaging strategies are still restricted by limited tumor specificity and sensitivity due to off-tumor signal leakage and a lower content of the targets. Therefore, a self-powered and self-recycled strategy based on molecular beacon (MB) hybridized on tetrahedral framework DNA (E-MB-tFNA) using the endogenous enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), was rationally developed for tumor cell-specific molecular imaging with superior spatial specificity and improved sensitivity. The apurinic/apyrimidinic site (AP site) can be specifically recognized and cleaved by APE1, and was modified on the stem and loop region of E-MB-tFNA. The resultant E-MB-tFNA can be specifically recognized and cleaved by APE1 in the cytoplasm, which is overexpressed in tumor cells compared to normal cells. In particular, once APE1 cleaves the AP site on the stem region (double-stranded DNA) of E-MB-tFNA, the self-powered and self-recycled sequences modified on the stem region of E-MB-tFNA is freed to hybridize with the loop region of E-MB-tFNA, prompting APE1 to further hydrolyze the E-MB-tFNA loop region and generate fluorescence signals. In addition, the self-powered and self-recycled sequences can be hybridized with other E-MB-tFNA to continuously and cyclically amplify the fluorescence signal. The experimental results of this study showed that the E-MB-tFNA has the capability to monitor drug resistance and achieve guided precision surgical excision in vivo with superior spatial specificity and improved sensitivity. In particular, E-MB-tFNA can realize the risk classification of neuroblastoma patients in clinical plasma.