Remarkable Tumor Regression Research Articles

Transforming Lung Cancer Management: A Promising Case Study of Immune Checkpoint Inhibitor Success Following a Multidisciplinary Approach

A 54-year-old female patient diagnosed with Stage IIIb squamous cell carcinoma (cT2aN3M0) initially received chemoradiotherapy. Two years after initial treatment, cancer relapse led to the administration of nivolumab, which was halted due to the development of drug-induced pneumonitis. Subsequent management with prednisolone and eight different cytotoxic agents failed to prevent metastasis to the cervical lymph nodes. The tumor’s programmed death-ligand 1 (PD-L1) expression rate was recorded at 10%. Four years after her diagnosis, the patient received a ninth-line therapy combining cisplatin, gemcitabine, and necitumumab, followed by palliative neck radiation due to increasing lymph node size. Remarkable tumor regression occurred three months after introducing atezolizumab as the tenth-line treatment, suggesting that previous treatments, particularly radiotherapy and cisplatin, might have enhanced PD-L1 expression, aligning with the existing literature. This case highlights the urgent need for further research to elucidate the intricate interplay between treatment history and PD-L1 expression in squamous cell carcinoma, emphasizing the importance of accumulating case studies to inform therapeutic strategies.

A novel B7-H3 x CD3 bispecific T-cell engager for the treatment of B7-H3+ tumors.

e14509 Background: B7-H3 (CD276) is a transmembrane protein highly expressed in solid tumors but with limited expression in normal tissues. Its presence is strongly associated with the inhibition of T-cell activity, cancer progression, and invasion. Furthermore, B7-H3 is strongly associated with poor prognosis and reduced patient survival. Given its crucial role in tumor biology, B7-H3 has emerged as an attractive therapeutic target for drug development. We utilized ExMab heterodimer platform and common light chain technology to develop EX105, a next-generation B7-H3×CD3 bispecific antibody. This innovative T-cell engager exhibits excellent anti-tumor activity in mouse models and good safety profile in NHP tox study. Methods: EX105 was generated using ExMab heterodimer platform and common light chain technology to form a heterodimeric IgG1 structure. Preclinical pharmacology studies, efficacy, and safety were evaluated. Anti-tumor activity study of EX105 includes in vitro T-cell cytotoxicity assays and in vivo PBMC reconstituted xenograft mice models. Preclinical safety evaluation of EX105 was performed through GLP single-dose and repeat-dose tox studies in non-human primates (NHPs). Results: Based on ELISA and FACS analyses, EX105 can bind to both human and cynomolgus monkey B7-H3 and CD3 antigens. Furthermore, SPR data suggest that EX105 exhibits a high affinity to B7-H3 and a much lower affinity to CD3. In consistent with that, EX105 only induced relatively low cytokine release. Nevertheless, EX105 showed a potent in vitro T cell cytotoxicity against B7-H3+ tumor cell lines. The in vivo anti-tumor activity of EX105 was further determined in several tumor xenograft models. Treatment with EX105 resulted in remarkable tumor regression in both “hot” tumor model and “cold” tumor model, indicating its effectiveness across a range of tumor types. Tox study in NHPs showed minimal adverse effects and no liver impairment after EX105 administration, even at high doses. The cytokine release data observed in this study suggests a good safety profile of EX105, with much lower cytokine release compared to other T cell engagers. Conclusions: These findings collectively indicate that EX105 has the potential to become a promising therapeutic agent for the treatment of various B7-H3 positive cancers. The first-in-human clinical trial for patients with advanced B7-H3 positive cancers is due to start.

Personalized Nanovaccines Enhance Lymph Node Accumulation and Reprogram the Tumor Microenvironment for Improved Photodynamic Immunotherapy.

Tumor immunotherapy has emerged as an efficacious therapeutic approach that mobilizes the patient's immune system to achieve durable tumor suppression. Here, we design a photodynamic therapy-motivated nanovaccine (Dex-HDL/ALA-Fe3O4) co-delivering 5-aminolevulinic acid and Fe3O4 nanozyme that demonstrate a long-term durable immunotherapy strategy. After vaccination, the nanovaccine exhibits obvious tumor site accumulation, lymph node homing, and specific and memory antitumor immunity evocation. Upon laser irradiation, Dex-HDL/ALA-Fe3O4 effectively generates reactive oxygen species at the tumor site not only to induce the immunogenic cell death-cascade but also to trigger the on-demand release of full types of tumor antigens. Intriguingly, Fe3O4 nanozyme-catalyzed hydrogen peroxide generated oxygen for alleviating tumor hypoxia and modifying the inhibitory tumor microenvironment, thereby exhibiting remarkable potential as a sensitizer. The intravenous administration of nanovaccines in diverse preclinical cancer models has demonstrated remarkable tumor regression and inhibition of postoperative tumor recurrence and metastasis, thereby enabling personalized treatment strategies against highly heterogeneous tumors.

Abstract 2963: Dihydromyricetin is a senomorphic agent to promote therapeutic outcome of age-related conditions including cancer and Alzheimer's disease

Abstract Aging is characterized by a progressive decline of organ function and gradual rise of susceptibility to multiple chronic disorders, with cancer being a primary cause of human morbidity and mortality worldwide. Cellular senescence represents a state of permanent cell cycle arrest and contributes to chronological aging and age-related diseases including Alzheimer's disease (AD), mainly through development of a distinct secretome termed the senescence-associated secretory phenotype (SASP). By screening a natural medicinal agent library, we noticed Rattan tea extract (RTE) has a prominent antiaging potential, wherein dihydromyricetin (DMY), the most abundant natural flavonoid in RTE, played a central role in suppressing the SASP expression of senescent cells. DMY enhanced total antioxidant capacity (T-AOC), increased catalase (CAT) and glutathione peroxidase (GSH-Px) activity, upregulated the expression of HO-1, NQO1, nuclear Nrf2 and phospho-ERK (p-ERK), but decreased malondialdehyde (MDA) level in senescent cells. Furthermore, DMY regulates PI3K/Akt/mTOR signaling pathway, NLRP3 inflammasome formation, the NF-κB complex activity and ER stress-response, each responsible for a full spectrum SASP development. Transcriptomic profiling suggested that DMY can dampen the genome-wide upregulation of pro-inflammatory factors produced by senescent cells, resulting in abrogated progression of cancer malignancy including elevated proliferation, migration, invasion and chemoresistance. Importantly, combination of DMY with classic chemotherapy significantly enhanced therapeutic efficacy, causing remarkable tumor regression and extending posttreatment survival. Alzheimer's disease (AD) is marked by the presence of amyloid plaques, neurofibrillary tangles, damaged neuronal macromolecules and redox sensitive ions. We found that DMY restrains inflammatory injury in microglial cells, reduces their apoptosis and inflammation, and inhibits TLR4 and MD2 expression. Results of small molecule-protein docking and pull-down assays indicated that DMY binds MD2 to suppress the formation of TLR4 complex. In AD mice, DMY improved the cognitive disorder of animals, suppressed inflammatory injury in brain and reduced TLR4 expression. Together, DMY represents a valid natural candidate that has senomorphic activity and can be applied for treatment of malignancies or neurodegenerative disorders such as AD, pathologies that frequently arise in late life of humans. Citation Format: Qixia Xu, Zi Li, Zhirui Jiang, Judith Campisi, Yu Sun. Dihydromyricetin is a senomorphic agent to promote therapeutic outcome of age-related conditions including cancer and Alzheimer's disease [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 2963.

Abstract 6059: Discovery of ETS-007, a first-in-class degrader targeting N-terminal domain of androgen receptor derived from AR liquid-liquid phase separation inhibitor

Abstract Patients with advanced prostate cancer inevitably acquire resistance to anti-androgen therapies, and progress to lethal castration-resistant prostate cancer (CRPC). The resistance mechanisms involve AR gene amplification and overexpression, constitutively active ligand-independent splice variants such as AR-V7, and the mutations in the AR ligand-binding domain (LBD). Our previous study has shown that the AR N-terminal domain (NTD) is responsible for AR liquid-liquid phase separation (LLPS) capability, which is essential for AR/AR-V7 transcriptional activity. We conducted a compound library screening using an AR LLPS phenotypic assay and identified ET0516 as a hit to specifically inhibit AR LLPS and its downstream gene expression, indicating that inhibition of AR. LLPS through targeting its NTD is a feasible approach to overcome the resistance to current anti-androgens therapy. Here we report the identification of a novel AR degrader, ETS-007 which is originated and evolved from internal LLPS phenotypic screening, that effectively degrades both AR and AR-V7 proteins with DC50 of single-digit nM by inducing a protein-protein interaction between AR NTD and an E3 ligase complex. ETS-007 induced AR and AR-V7 polyubiquitination, followed by degradation which could be blocked by the protease inhibitor MG132. ETS-007 significantly inhibited cell proliferation in AR and/or AR-V7 positive cell lines, e.g. LNCaP and 22RV1, and downregulated the androgen responsive genes such as PSA and TMPRSS2. Compared with enzalutamide and ARV-110, ETS-007 could block transcriptional activities of AR-V7, and AR mutants in luciferase report assay. Oral administration of ETS-007 in an enzalutamide-resistant 22RV1 xenograft model led to intratumoral AR-V7 degradation and concomitant decrease in PSA protein levels, resulting in a remarkable tumor regression. Taken together, our findings provide preclinical evidence that ETS-007 as a potent AR-NTD degrader derived from our LLPS platform can effectively block the transcriptional activities of both AR and AR-V7 for the treatment of metastatic CRPC. Citation Format: Yingjie Li, Mingyue Fei, Yingke Miao, Lijian Feng, Xiaofei Fan, Jinping Li, Yaozhong Chen, Ming Xu, Wenqi Cui, Qiangang Zheng, Jidong Zhu. Discovery of ETS-007, a first-in-class degrader targeting N-terminal domain of androgen receptor derived from AR liquid-liquid phase separation inhibitor [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 6059.

Abstract 1938: WEE1 inhibition prevents and overcomes resistance to KRAS inhibitors in lung cancer by enhancing MCL1-mediated apoptosis

Abstract Background: While novel covalent inhibitors of KRAS G12C have been approved in non-small-cell lung cancer (NSCLC), their efficacies are known to be limited. To enhance the therapeutic potential of KRAS inhibitors, combination strategies need to be developed. In this study, we conducted a large-scale screening to identify novel combination partners that induced synergistic efficacy with KRAS inhibitors. Additionally, mechanistic analyses were conducted to clarify the synergistic effects in combination and their role in conferring resistance to KRAS inhibitors. Methods: To identify novel combination partners of KRAS inhibitors, we conducted high-throughput screening (HTS) using a chemical library composed of 1,400 kinase inhibitors. Sotorasib was used as a KRAS inhibitor. Anti-proliferative and apoptotic effects in vitro were assessed by ATP-based cell viability assay, and caspase 3/7 assay and/or Annexin V assay, respectively. Protein expression levels were assessed by immunoblotting. In vivo studies were performed using a H358 xenograft nude mouse model and a patient-derived xenograft model (PDX). PDX samples were provided by the National Cancer Center J-PDX library, Japan. Results: HTS identified the WEE1 inhibitor AZD1775, a G2/M checkpoint abrogator in clinical-stage development, as a promising combination partner of KRAS inhibitors in KRAS G12C mutant NSCLC. Synergistic effects of KRAS and WEE1 inhibitors were confirmed across multiple KRAS G12C mutant NSCLC cell lines, independently of their co-occurring oncogenic mutation profiles. The combination treatment enhanced apoptotic cell death, being upregulated pro-apoptotic protein BIM, while downregulated anti-apoptotic protein MCL1. In vivo efficacy studies in combination with sotorasib and AZD1775 also demonstrated remarkable tumor regression and durable response in both CDX and PDX models. We also generated sotorasib-resistant H23 (H23-SR) cells, confirming upregulation of both WEE1 and MCL1 in the resistant cells. Ectopic overexpression of WEE1 in H23, on the contrary, conferred resistance to sotorasib. Furthermore, WEE1 inhibition re-sensitized H23-SR cells to KRAS inhibitors. Conclusion: Our results suggest that the WEE1-MCL1 axis plays an important role in both the initial therapeutic efficacy and the acquired resistance to KRAS inhibitors. These findings could pave the way for a novel therapeutic strategy for KRAS G12C mutant NSCLC. Citation Format: Gaku Yamamoto, Kosuke Tanaka, Ryo Kamata, Takehiro Nakao, Shunta Mori, Jie Liu, Susumu S. Kobayashi, Akihiro Ohashi. WEE1 inhibition prevents and overcomes resistance to KRAS inhibitors in lung cancer by enhancing MCL1-mediated apoptosis [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 1938.

Design, synthesis and antitumor activity of 4-arylamine substituted pyrimidine derivatives as noncovalent EGFR inhibitors overcoming C797S mutation

Clinical researches have shown that epidermal growth factor receptor (EGFR) is a key target for treatment of non-small cell lung cancer (NSCLC). Many EGFR inhibitors were successfully developed as ani-tumor drugs to treat NSCLC patients. Unfortunately, drug resistances were found in clinic. To overcome C797S mutation in EGFR, a novel series of 4-arylamine substituted pyrimidine derivatives were designed and synthesized under the principle of structure-based drug design. Interestingly, compounds 6e and 9i demonstrated the best anti-proliferative activity against A549, NCI–H1975, and HCC827 cells. In particular, the IC50 values against HCC827 cells reached to 24.6 nM and 31.6 nM, which were much lower than human normal cells 2BS and LO2. Furthermore, compounds 6e and 9i showed extraordinary activity against EGFR19del/T790M/C797S (IC50 = 16.06 nM and 37.95 nM) and EGFRL858R/T790M/C797S (IC50 = 11.81 nM and 26.68 nM), which were potent than Osimertinib (IC50 = 52.28 nM and 157.60 nM). Further studies have shown that compounds 6e and 9i could pertain inhibition of HCC827 colony formation, and arrest HCC827 cells at G2/M phase. Moreover, the most promising compound 6e could inhibit the migration of HCC827 cells, induce HCC827 cells apoptosis, and significantly inhibit the phosphorylation of EGFR, AKT and Erk1/2. In vivo xenograft mouse model with HCC827 cells, compound 6e resulted in remarkable tumor regression without obvious toxicity. In addition, molecular docking studies suggested that compound 6e could firmly combine with T790M-mutant, T790 M/C797S-mutant, and L858R/T790 M/C797S-mutant EGFR kinases as ATP-competitive inhibitor.

Abstract B028: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a poorly immunogenic, high-grade neuroendocrine carcinoma arising in the lung. Immune checkpoint blockade (ICB) added to chemotherapy is now the standard upfront therapy for SCLC but leads to a modest increase in overall survival and progression-free survival. These modest benefits underscore the critical need to identify pathways and targets that can durably enhance the antitumor responses of ICB in SCLC. SCLC is characterized by a ubiquitous loss of TP53 which disrupts the G1-S cell cycle checkpoint and as a result, most SCLC is dependent on G2-M cell cycle checkpoint regulators including WEE1. We and others have shown that DNA damage response (DDR) components are overexpressed in SCLC and targeting DDR could be an effective therapeutic strategy in SCLC. Methods: In this study, we have performed WEE1 inhibition either alone or in combination with PD-L1 blockade in a panel of SCLC models. Results: We demonstrate that inhibition of WEE1 induces G2/M cell cycle arrest, DNA damage, and cytosolic DNA accumulation in SCLC models. We further show that WEE1 targeting activates the STING-TBK1-IRF3 pathway which increases type I interferons (IFN-α and IFN-β) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T-cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade caused remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T-cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Conclusions: Our study demonstrates the cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC. Given the increasing importance of immunotherapy for the management of SCLC and that WEE1 inhibitors are already in clinical trials, combining a WEE1 inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B028.

Intelligent Responsive Nanoparticles with Multilevel Triggered Drug Penetration for Tumor Photochemotherapy.

Nanomedicines have contradictory size requirements to overcome systemic barriers and penetrate the tumor extracellular matrix (ECM). Larger-sized nanoparticles (50-200 nm) exhibit prolonged blood circulation half-life and improved tumor enrichment, while small-sized nanoparticles (4-20 nm) easily penetrate deep tumor tissues. Therefore, the development of intelligent responsive nanomedicine systems can not only increase nanodrug tumor accumulation but also improve their penetration into the ECM. Herein, we propose an intelligent responsive nanoparticle triggered by near-infrared light (NIR). The nanoparticle was constructed by a temperature-sensitive liposome (TSL) encapsulating ultrasmall melanin nanoparticles (MNPs) loaded with doxorubicin (MNP/doxorubicin (DOX)@TSL). When exposed to NIR irradiation, the tailor-made nanoparticles not only effectively ablated the tumor cells around blood vessels but also destroyed the structural integrity and released loaded ultrasmall MNP/DOX (<10 nm) to promote deep tumor penetration and enhance interior tumor cell killing. This NIR-triggered intelligent nanoparticle successfully integrated photothermal therapy (PTT) for perivascular tumor cells and chemotherapy for deep tumor cell inhibition. The in vivo results showed remarkable tumor regression in 4T1 breast tumor-bearing mice by 74.2%. This controllable size switchable nanosystem with efficient tumor accumulation and penetration has shown great potential in improving synergistic antitumor effects of photochemotherapy.

PH-sensitive liposomes bearing a chemotherapeutic agent and a natural apoptosis modulator for effective intracellular delivery to the solid tumor.

The intracellular delivery of the drug to the solid tumor is a major challenge in the treatment of solid tumors. This project aims to increase cytosolic drug delivery using the endosomal escape of drugs. Topotecan (TPT) and capsaicin were used for the treatment of solid tumors. The pH-dependent conversion of active lactone form to inactive carboxylic form is a major problem of TPT that limits its therapeutic use. Liposomal encapsulation of TPT improved the stability of active lactone form and increased the therapeutic efficacy of TPT. Endosomal degradation of liposomes may reduce the content in the target cells. To solve these problems, pH-sensitive liposomes (pSLPs) were developed which improved the intracellular drug delivery by the endosomal escape of drugs. The liposomes (LPs) bearing the drug(s) were prepared using the cast film method and optimized for various formulation and process variables using the Design-Expert 7 software by employing the Box-Behnken design (BBD). The developed hyaluronic acid (HA)-conjugated pSLPs (HA-pSLPs) displayed a vesicle size of 166.5 ± 2.31nm, zeta potential - 30.53 ± 0.91, and entrapment efficiency of 44.39 ± 1.78%, and 73.48 ± 2.15% for TPT and CAP, respectively. HA-pSLPs displayed better cytotoxicity in comparison to free drugs either single or in combination on the MCF-7 cell line. The apoptosis and cellular uptake of HA-pSLPs were increased ⁓ 4.45-fold and ⁓ 6.95-fold as compared to unconjugated pSLPs, respectively. The pharmacokinetic studies in Balb/c mice demonstrated that HA-pSLPs increased the half-life, MRT, and AUC in comparison to the free drug solution. The HA-pSLPs formulation has shown remarkable tumor regression as compared to PpSLPs, pSLPs, and free drug combinations. These results demonstrated that TPT- and CAP-loaded HA-pSLPs offer a potential platform for targeted drug delivery to solid tumors.

Abstract 5977: Evaluation of the potential combination regimens for q901, a clinical stage selective cdk7 inhibitor, as a DNA damage repair inhibitor

Abstract Background; Q901 is an extremely selective CDK7 inhibitor with potent single agent activity in the multiple in vivo tumor models including small cell lung cancer, cholangiocarcinoma, colon cancer, pancreatic cancer, hormone receptor positive breast cancer, castrate-resistant prostate cancer, and ovarian cancer. Q901 is currently in Phase 1/2 clinical trial for patients with selected advanced solid tumors (ClinicalTrials.gov: NCT05394103). In the preclinical models, Q901 treatment induces DNA damage response (DDR) impairment markers, particularly ones involved in the double-strand break repair mechanisms. This opens up broad range of possibilities for new combination regimen that could benefit from the accumulated DNA damages caused by Q901 treatment. Methods: Various combinations have been tested to evaluate potential clinical regimens in multiple solid tumor models. Two different BRCA wild-type ovarian cancer A2780 and OVCAR3 models, an estrogen receptor positive MCF7 breast cancer model, or a syngeneic RENCA mouse renal cancer model was tested with Q901 in combination with poly ADP-ribose polymerase (PARP) inhibitor, selective estrogen receptor degrader (SERD), an estrogen receptor (ER) PROTAC degrader, or an anti-PD-1 antibody, respectively. Results: In the A2780 model, the PARPi alone group showed 15% TGI (tumor growth inhibition), but significant tumor regression (104% TGI) was observed in the Q901 and PARPi combination. In the OVCAR3 model, the Q901 and PARPi combination group showed 105% TGI, whereas the PARPi alone group showed 38% TGI. In the MCF7 model, SERD alone showed 64% TGI and Q901 combination with SERD showed 94% TGI. Remarkable tumor regression (111% TGI) was observed in the MCF7 model combining ER PROTAC and Q901, whereas ER PROTAC alone group showed 55% TGI. In the RENCA model, the anti-PD-1 antibody alone group showed 13.2% TGI and the combination of Q901 and anti-PD-1 antibody showed 66% TGI. In summary, these preclinical model studies demonstrated significant additive/synergistic effects of the Q901 combination on tumor growth inhibition. Conclusion: The non-clinical data demonstrate that Q901 not only has a potential as a single agent,but can also be used in combination with other therapeutic strategies to develop next-generation cancer therapeutics to potentially address unmet medical needs. Citation Format: Donghoon Yu, Yeejin Jeon, YoonJi Lee, Seung-Joo Lee, Jaeseung Kim, Hyerim Jung, Tae-Kyung Kim, Kiyean Nam. Evaluation of the potential combination regimens for q901, a clinical stage selective cdk7 inhibitor, as a DNA damage repair inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5977.

Abstract 5739: Targeting WEE1 to improve the therapy of KRAS G12C mutant non-small cell lung cancer

Abstract Background: While sotorasib is the first clinically approved small-molecule inhibitor of KRAS G12C in non-small-cell lung cancer (NSCLC), efficacy of sotorasib alone is limited, presumably due to adaptive resistance mechanisms in cancer cells. To expand therapeutic potential of sotorasib, combination strategies need to be developed. In this study, we conducted a large-scale screening to identify novel combination candidates with sotorasib. In addition, molecular mechanisms of synergistic effects were investigated in multiple preclinical models. Methods: To identify novel combination partners of sotorasib, we conducted high-throughput screening using a kinase inhibitor library containing 1,400 chemical compounds. Anti-proliferative and apoptotic effects in vitro were assessed by ATP-based cell viability assay and/or clonogenic assay, and caspase 3/7 assay and/or Annexin V assay, respectively. Synergistic effects were evaluated by calculating the BLISS index and visualized by SynergyFinder Plus. Immunoblotting was performed using antibodies against pCDK1 (Tyr15), pERK, pAkt, MCL1, BIM, PUMA, BCL2, or BCL-xL. In vivo efficacy studies were performed using a KRAS G12C mutant H358 xenograft nude mouse model. Results: Through the large-scale unbiased combination screening of -1,400 kinase inhibitors, we identified a WEE1 inhibitor (AZD1775), a G2/M checkpoint abrogator in clinical-stage development, as a promising combination partner of sotorasib in KRAS G12C mutant NSCLC. The synergistic effects in vitro were broadly observed in multiple KRAS G12C mutant NSCLC cell lines, regardless of single-agent sensitivity to sotrasib or co-occurring oncogenic mutation profiles. The combination treatment upregulated pro-apoptotic protein BIM, leading to apoptosis. In vivo efficacy studies in H358-xenografted models also demonstrated remarkable tumor regression and durable response in mice treated with sotorasib and AZD1775 compared to ones with a single agent alone. Conclusion: We identify AZD1775, a WEE1 inhibitor, as a novel combination candidate, which enhanced the anti-tumor activity of sotorasib both in vitro and in vivo preclinical models. These findings can lead to a novel therapeutic strategy for KRAS G12C mutant NSCLC. Citation Format: Gaku Yamamoto, Kosuke Tanaka, Ryo Kamata, Shunta Mori, Jie Liu, Toyohiro Yamauchi, Yuta Sakae, Akihiro Ohashi, Susumu S. Kobayashi. Targeting WEE1 to improve the therapy of KRAS G12C mutant non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5739.

Förster Resonance Energy Transfer Nanobullet for Photoacoustic Imaging and Amplified Photothermal-Photodynamic Therapy of Cancer.

Synergistic photodynamic and photothermal therapy (PDT-PTT) has emerged as an appealing effective antitumor approach. However, clinical utilization of PDT-PTT is plagued by aggregation-caused photobleaching, sequential double irradiations, unsatisfying balance between single oxygen (1 O2 ) quantum yield and photothermal conversion efficiency. Here, an anchored tumor-homing cell-penetrating peptide (PEGA-pVEC) and PANI-ES/HMME loaded FRET nanobullet (AHP-P) are reported. Within nanobullet, HMME (donor) and PANI-ES (acceptor) spontaneously form a förster resonance energy transfer (FRET) pair. Upon 660nm laser irradiation, HMME convert near-infrared fluorescence (NIRF) to PANI, thus produce FRET-amplified photoacoustic imaging guided PTT. In addition, AHP-P with pH-sensitivity can gradually release HMME within acidic tumor environment, boosts the 1 O2 regeneration alongside with highly efficient photothermal conversion for photoinduced cancer PTT-PDT. Furthermore, the AHP-P nanobullet can home in on the tumor site and penetrate into cytoplasm through PEGA-pVEC, inducing remarkable tumor regression with an ≈80% tumor volume reduction and decreased skin phototoxicity in vivo during FRET-amplified PTT-PDT.

Photoactivatable nanoagonists chemically programmed for pharmacokinetic tuning and in situ cancer vaccination.

Immunotherapy holds great promise for the treatment of aggressive and metastatic cancers; however, currently available immunotherapeutics, such as immune checkpoint blockade, benefit only a small subset of patients. A photoactivatable toll-like receptor 7/8 (TLR7/8) nanoagonist (PNA) system that imparts near-infrared (NIR) light-induced immunogenic cell death (ICD) in dying tumor cells in synchrony with the spontaneous release of a potent immunoadjuvant is developed here. The PNA consists of polymer-derived proimmunoadjuvants ligated via a reactive oxygen species (ROS)-cleavable linker and polymer-derived photosensitizers, which are further encapsulated in amphiphilic matrices for systemic injection. In particular, conjugation of the TLR7/8 agonist resiquimod to biodegradable macromolecular moieties with different molecular weights enabled pharmacokinetic tuning of small-molecule agonists and optimized delivery efficiency in mice. Upon NIR photoirradiation, PNA effectively generated ROS not only to ablate tumors and induce the ICD cascade but also to trigger the on-demand release of TLR agonists. In several preclinical cancer models, intravenous PNA administration followed by NIR tumor irradiation resulted in remarkable tumor regression and suppressed postsurgical tumor recurrence and metastasis. Furthermore, this treatment profoundly shifted the tumor immune landscape to a tumoricidal one, eliciting robust tumor-specific T cell priming invivo. This work highlights a simple and cost-effective approach to generate insitu cancer vaccines for synergistic photodynamic immunotherapy of metastatic cancers.

Combined Targeted Radiopharmaceutical Therapy and Immune Checkpoint Blockade: From Preclinical Advances to the Clinic.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer care, but many patients with poorly immunogenic tumors fail to benefit. Preclinical studies have shown that external beam radiotherapy (EBRT) can synergize with ICI to prompt remarkable tumor regression and even eradication. However, EBRT is poorly suited to widely disseminated disease. Targeted radiopharmaceutical therapy (TRT) selectively delivers radiation to both the primary tumor and the metastatic sites, and promising results achieved with this approach have led to regulatory approval of certain agents (e.g., 177Lu-PSMA-617/Pluvicto for metastatic prostate cancer). To further improve therapeutic outcomes, combining TRT and ICI is a burgeoning research area, both preclinically and in clinical trials. Here we introduce basic TRT radiobiology and survey emerging and clinically translated TRT agents that have been combined with ICI.

WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC.

SUMMARYSmall cell lung cancers (SCLCs) have high mutational burden but are relatively unresponsive to immune checkpoint blockade (ICB). Using SCLC models, we demonstrate that inhibition of WEE1, a G2/M checkpoint regulator induced by DNA damage, activates the STING-TBK1-IRF3 pathway, which increases type I interferons (IFN-α and IFN-β) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade causes remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Our study demonstrates cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC models. Combined inhibition of WEE1 plus PD-L1 blockade represents a promising immunotherapeutic approach in SCLC.

Improving Cancer Detection and Treatment by pH‐Sensitive Peptide Nanoparticle Drug Delivery Platform: Pharmacokinetics, Toxicity, and Immunogenicity Profile

It was previously reported that a nanoparticle made of ultra pH‐sensitive peptides (pH‐NP) undergoes self‐dissolution in moderately acidic (pH ≤ 6.9) condition that mimics the pH in extracellular tumor environment, leading to release of drug cargo molecules. Herein, it is demonstrated that this peptide‐based nanodelivery platform can be used to formulate poorly water‐soluble small molecules to improve cancer diagnosis and treatment. Indocyanine green (ICG) encapsulated in pH‐NP (ICG‐pH‐NP) significantly increases tumor‐specific accumulation compared with “free” ICG, leading to excellent in vivo optical imaging contrast. An investigational cancer drug, OTS964, formulated in pH‐NP (OTS‐pH‐NP), sensitizes the drug‐resistant triple‐negative breast cancer to chemotherapy (paclitaxel), resulting in a remarkable tumor regression, whereas OTS964 in standard formulation does not (P = 0.0004). The potential immunogenicity and bone marrow toxicity of this nanoparticle are further investigated by administering drug‐free pH‐NP at nanoparticle dose regimens, representing 1.5× and 4.5×, respectively, of which are used in the treatment (OTS‐pH‐NP) study. The data show that the pH‐NP is not immunogenic and is well‐tolerated. Taken together, self‐dissolution and the release of drug cargo in response to the tumor's acidic extracellular pH faciliates drug delivery when mediated by the pH‐NP nano platform, making it a promising, clinical translational delivery platform for enhancing cancer detection and treatment.

Evaluation of Hedgehog Pathway Inhibitors as a Therapeutic Option for Uterine Leiomyosarcoma Using the Xenograft Model

Uterine leiomyosarcoma (LMS) contributes to a significant proportion of uterine cancer deaths. It is a rare and high-risk gynecological cancer. LMS is challenging to the treatment due to the resistance of several therapies. The activation of the Hedgehog (HH) pathway has been reported in several types of female cancers. Uterine LMS presents an upregulation of the crucial HH signaling pathway members such as SMO and GLI1. Although targeting the HH pathway exhibited a potent inhibitory effect on the phenotype of uterine LMS in vitro, the effect of the HH inhibitors on LMS growth in vivo has not been identified. The present study aimed to assess the effect of Hedgehog pathway inhibitors (SMO-LDE225 and GLI-Gant61) as a therapeutic option in the xenograft model of uterine LMS. The results demonstrated that LDE225 treatment did not show any inhibitory effect on LMS tumor growth; however, treatment with GLI inhibitor (Gant61) induced a remarkable tumor regression with a significant decrease in Ki67 expression, compared to control (p < 0.01). Moreover, administration of Gant61 decreased the expression of GLI1, GLI target genes BMP4 and c-MYC (p < 0.05), indicating that the HH pathway is implicated in the LMS experimental model. In conclusion, our studies demonstrate for the first time that GLI inhibitor (Gant61), but not SMO inhibitor (LDE225), shows a potent inhibitory effect on LMS tumor growth and concomitantly suppresses the expression of GLI1- and GLI-targeted genes using the xenograft model of uterine LMS.

EVALUATION OF HEDGEHOG PATHWAY INHIBITORS AS A THERAPEUTIC OPTION FOR UTERINE LEIOMYOSARCOMA USING THE XENOGRAFT MODEL

Uterine leiomyosarcoma (LMS) contributes to a significant proportion of uterine cancer deaths. It is a rare and high-risk gynecological cancer. LMS is challenging to the treatment due to the resistance of several therapies. The activation of the Hedgehog (HH) pathway has been reported in several types of female cancers. Uterine LMS presents an upregulation of the crucial HH signaling pathway members such as SMO and GLI1. Although targeting the HH pathway exhibited a potent inhibitory effect on the phenotype of uterine LMS in vitro, the effect of the HH inhibitors on LMS growth in vivo has not been identified. The present study aimed to assess the effect of Hedgehog pathway inhibitors (SMO-LDE225 and GLI-Gant61) as a therapeutic option in the xenograft model of uterine LMS. The results demonstrated that LDE225 treatment did not show any inhibitory effect on LMS tumor growth; however, treatment with GLI inhibitor (Gant61) induced a remarkable tumor regression with a significant decrease in Ki67 expression, compared to control (p < 0.01). Moreover, administration of Gant61 decreased the expression of GLI1, GLI target genes BMP4 and c-MYC (p < 0.05), indicating that the HH pathway is implicated in the LMS experimental model. In conclusion, our studies demonstrate for the first time that GLI inhibitor (Gant61), but not SMO inhibitor (LDE225), shows a potent inhibitory effect on LMS tumor growth and concomitantly suppresses the expression of GLI1- and GLI-targeted genes using the xenograft model of uterine LMS.

Abstract 439: BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer

Abstract BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer Background: Immune checkpoint inhibitors (ICIs) has received approval due to its encouraging clinical efficacy with improving the outcome of cancer patients. However, current immunotherapeutic regimens benefit merely a minority of populations. Thus, combining ICIs with other potential modalities is of great significance to enhance the response of single drug alone. Modifying tumor-associated macrophages (TAMs) strategies have attracted attention for ameliorating tumor immunosuppression and improving anti-tumor immunity. Here we exhibit APG2575, a proven BCL-2 inhibitor, can act as an antitumor immune modulator that resets TAMs from M2 to pro-tumoral M1 phenotype and further augment immunotherapeutic efficacy. Methods: By applying non-small cell lung cancer cell lines and murine syngeneic tumor xenograft, we investigated the effects of combined APG-2575 and PD-1 antibody on tumor growth. Furthermore, we explored the role of APG-2575 in regulating TAMs polarization through utilizing human and murine macrophages, involving immortalized and primary models. Besides, IL-10 secretion in cell culture supernatants was detected via ELISA assay. In addition, we also assessed the tumor immune microenvironment regulation of APG-2575 using flow cytometry. Results: In vitro, APG-2575, a BCL-2 inhibitor, augmented the tumor cell lysis induced by T cells when combined with ICIs. Moreover, mice treated with APG-2575 and ICIs showed remarkable tumor regression and prolonging survival which were associated with the increase of granzyme-B, IFN-γ and TNF-α release of CD8+ cytotoxic lymphocytes. Importantly, we found that APG-2575-mediated antitumor T cell immunity is dependent on macrophages. Furthermore, we discovered APG-2575 could induce M2 macrophage switching to M1 phenotype and suppress IL-10 secretion in vitro. Meanwhile, reduced M2 macrophages as well as increased M1 polarization were also observed in the tumor-infiltrating immune cells from mice treated with APG-2575. Additionally, APG-2575-mediated macrophage transition could improve tumor immunosuppression, with decreasing number of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells in tumor microenvironment. Conclusions: Our data demonstrate that APG-2575 can synergizes with ICIs in vitro and in vivo, which was involved in switching TAMs from M2 to M1 polarization and further improving tumor immunosuppression, finally enhancing T cell anti-tumor activity. These results warranted future clinical evaluation of combination therapy of APG-2575 and ICIs. Key words: BCL-2, APG-2575, ICIs, macrophages, non-small cell lung cancer Citation Format: Fan Luo, Fei Teng Lu, Miao Zhen Qiu, Wen Tao Pan, Lin Zhang, Hong Yun Zhao, Li Zhang, Da Jun yang. BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 439.