Patient-derived Xenograft Mouse Models Research Articles

A molecular glue RBM39-degrader induces synthetic lethality in cancer cells with homologous recombination repair deficiency

E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.

Abstract IA019: Lessons from glioblastoma diversity, heterogeneity, and beginnings

Abstract Glioblastoma (GBM) is a dire disease with poor prognosis and survival outcomes have changed little over the past two decades. Various genomic studies have emphasized the heterogeneity of GBM yet a consensus on the causes and its role in tumor recurrence has not been reached. Multiple studies using genetic and patient-derived xenograft (PDX) mouse models have demonstrated an inverse relationship between differentiation state and tumor initiation efficiency with neural stem and progenitor cells as the most efficient. However, other studies have proposed that well differentiated cells like neurons and astrocytes can form tumors. In addition, bioinformatic analyses of human primary GBM have reported that plasticity allows for mobility among cell states exist. Regardless of transcriptional modulation in GBM cells, to achieve progress in understanding and addressing clinical recurrence, the outstanding goal remains to identify and isolate the GBM cell type(s) responsible for therapy resistance and recurrence. To achieve this, we have employer autochthonous GBM models as well as orthotopic patient derived xenografts to in parallel examine tumor heterogeneity, cell of origin, and functional consequences. Citation Format: Luis F. Parada. Lessons from glioblastoma diversity, heterogeneity, and beginnings [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA019.

Abstract PO2-04-14: Dual Inhibition of WEE1 and PKMYT1 Synergistically Overcomes CDK4/6 Inhibitor Resistance in Breast Cancer

Abstract Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are approved for the treatment of advanced estrogen receptor-positive, HER2-negative (ER+ve) breast cancer when combined with endocrine therapy. However, the emergence of resistance to CDK4/6 inhibitors poses a significant clinical challenge. This study investigates the mechanisms underlying CDK4/6 inhibitor resistance and identifies a common occurrence of high replication stress in triple-negative breast cancer (TNBC) and CDK4/6 inhibitor-resistant ER+ve breast cancers. While ER+ve breast cancer patients have benefited from targeted therapy, TNBC patients, particularly those without germline BRCA1/2 or PALB2 mutations, lack effective treatment options. TNBC and CDK4/6 inhibitor-resistant ER+ve breast cancer cells exhibit an increased dependence on cell cycle arrest to repair replication stress-induced DNA damage. Failure to exit the cell cycle results in excessive DNA damage, thereby limiting their proliferative potential. Based on these findings, we hypothesize that dual inhibition of WEE1 and PKMYT1, two kinases involved in G2/M cell cycle transition and crucial for cell cycle arrest and DNA damage repair, could synergistically inhibit tumor growth. Although WEE1 and PKMYT1 share a common function in promoting G2/M cell cycle transition through CDK1 phosphorylation, their functions are not entirely redundant, suggesting that targeting both kinases may elicit a synergistic response. Recent clinical trials have shown resistance to WEE1-targeted monotherapies, emphasizing the need for combining WEE1 inhibitors with other agents, such as PKMYT1 inhibitors. We demonstrate the synergistic effects of WEE1 inhibitor (MK1775/AZD1775) and PKMYT1 inhibitor (RP6306) in a panel of TNBC and ER+ve breast cancer cells with acquired palbociclib resistance (PalboR). Combination treatment with both inhibitors leads to excessive unrepaired DNA damage, as indicated by increased γH2AX foci following 48 hours of combination treatment, whereas single-agent treatments induce significantly lower levels of DNA damage. Furthermore, we observed the AZD1775/RP6306 combination decreases the expression of the DNA repair protein Rad51 in PalboR cells. To evaluate the clinical potential of dual WEE1 and PKMYT1 inhibition, we generated organoids from metastatic breast cancer lesions of patients who had progressed on CDK4/6 inhibitors for ex vivo studies. Our results demonstrate that dual inhibition of WEE1 and PKMYT1 is more effective than single-agent treatments in eliminating CDK4/6 inhibitor-resistant organoids. Additionally, using patient-derived xenograft mouse models from patients who had progressed on palbociclib and endocrine treatment, we show that the AZD1775/RP6306 combination exhibits superior tumor suppression effects compared to single agents. Overall, this study highlights the potential therapeutic benefits of dual inhibition of WEE1 and PKMYT1 in overcoming CDK4/6 inhibitor resistance in TNBC and ER+ve breast cancer patients. These findings provide a rationale for future clinical trials aimed at exploring the clinical utility of this combination therapy. Citation Format: Mei-Kuang Chen, Grace DeAlessandro, Tuyen Bui, Khandan Keyomarsi. Dual Inhibition of WEE1 and PKMYT1 Synergistically Overcomes CDK4/6 Inhibitor Resistance in 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 PO2-04-14.

Patient tissue-derived FGFR4-variant and wild-type colorectal cancer organoid development and anticancer drug sensitivity testing

ObjectivesFGFR4-variant and wild-type colorectal cancer (CRC) organoids were developed to investigate the effects of FGFR4-targeted drugs, including FGFR4-IN and erdafitinib, on CRC and their possible molecular mechanism. MethodsClinical CRC tissues were collected, seven CRC organoids were developed, and whole exome sequencing (WES) was performed. CRC organoids were cultured and organoid drug sensitivity studies were conducted. Finally, an FGFR4-variant (no wild-type) CRC patient-derived orthotopic xenograft mouse model was developed. Western blot measured ERK/AKT/STAT3 pathway-related protein levels. ResultsWES results revealed the presence of FGFR4-variants in 5 of the 7 CRC organoids. The structural organization and integrity of organoids were significantly altered under the influence of targeted drugs (FGFR4-IN-1 and erdafitinib). The effects of FGFR4 targeted drugs were not selective for FGFR4 genotypes. FGFR4-IN-1 and erdafitinib significantly reduced the growth, diameter, and Adenosine Triphosphate (ATP) activity of organoids. Furthermore, chemotherapeutic drugs, including 5-fluorouracil and cisplatin, inhibited FGFR4-variant and wild-type CRC organoid activity. Moreover, the tumor volume of mice was significantly reduced at week 6, and p-ERK1/2, p-AKT, and p-STAT3 levels were down-regulated following FGFR4-IN-1 and erdafitinib treatment. ConclusionsFGFR4-targeted and chemotherapeutic drugs inhibited the activity of FGFR4-variant and wild-type CRC organoids, and targeted drugs were more effective than chemotherapeutic drugs at the same concentration. Additionally, FGFR4 inhibitors hindered tumorigenesis in FGFR4-variant CRC organoids through ERK1/2, AKT, and STAT3 pathways. However, no wild-type control was tested in this experiment, which need further confirmation in the next study.

The Synergy of 177Lu-FAPI-046 with Tyrosine Kinase Inhibitor in a Sarcoma Patient-Derived Xenograft Mouse Model

BackgroundGiven the heterogeneity and high mortality associated with metastatic soft tissue sarcoma, this study aims to evaluate the therapeutic efficacy of combining 177Lu-FAPI-46 with Pazopanib against this malignancy. MethodsPatient-derived xenograft (PDX)-bearing mice were randomly divided into three groups: the control group, the 177Lu-FAPI-46 monotherapy group, and the 177Lu-FAPI-46 combined with Pazopanib therapy group. Therapeutic efficacy was regularly monitored. ResultsThe microPET imaging showed a 0.84-fold decrease in the T/M ratio of 68Ga-FAPI-46 on day 7/8 post combination therapy, while the control group exhibited a 1.23-fold increase. Combination therapy significantly inhibited tumor proliferation, as evidenced by reduced Ki-67 and increased caspase 3 expressions. Notably, there was no significant body weight loss observed in any group. ConclusionThis study successfully demonstrated the reduction in FAP expression and suppression of tumor volume in sarcoma PDX following the combination therapy of 177Lu-FAPI-46 with Pazopanib.

Targeting leucine-rich PPR motif-containing protein/LRPPRC by 5,7,4′-trimethoxyflavone suppresses esophageal squamous cell carcinoma progression

JAK2/STAT3/MYC axis is dysregulated in nearly 70 % of human cancers, but targeting this pathway therapeutically remains a big challenge in cancer therapy. In this study, genes associated with JAK2, STAT3, and MYC were analyzed, and potential target genes were selected. Leucine-rich PPR motif-containing protein (LRPPRC) whose function and regulation are not fully understood, emerged as one of top 3 genes in terms of RNA epigenetic modification. Here, we demonstrate LRPPRC may be an independent prognostic indicator besides JAK2, STAT3, and MYC. Mechanistically, LRPPRC impairs N6-methyladenosine (m6A) modification of JAK2, STAT3, and MYC to facilitate nuclear mRNA export and expression. Meanwhile, excess LRPPRC act as a scaffold protein binding to JAK2 and STAT3 to enhance stability of JAK2-STAT3 complex, thereby facilitating JAK2/STAT3/MYC axis activation to promote esophageal squamous cell carcinoma (ESCC) progression. Furthermore, 5,7,4′-trimethoxyflavone was verified to bind to LRPPRC, STAT3, and CDK1, dissociating LRPPRC-JAK2-STAT3 and JAK2-STAT3-CDK1 interaction, leading to impaired tumorigenesis in 4-Nitroquinoline N-oxide induced ESCC mouse models and suppressed tumor growth in ESCC patient derived xenograft mouse models. In summary, this study suggests regulation of m6A modification by LRPPRC, and identifies a novel triplex target compound, suggesting that targeting LRPPRC-mediated JAK2/STAT3/MYC axis may overcome JAK2/STAT3/MYC dependent tumor therapeutic dilemma.

Oncogenic functions and therapeutic potentials of targeted inhibition of SMARCAL1 in small cell lung cancer

Small cell lung cancer (SCLC) is a recalcitrant cancer characterized by high frequency loss-of-function mutations in tumor suppressors with a lack of targeted therapy due to absence of high frequency gain-of-function abnormalities in oncogenes. SMARCAL1 is a member of the ATP-dependent chromatin remodeling protein SNF2 family that plays critical roles in DNA damage repair and genome stability maintenance. Here, we showed that SMARCAL1 was overexpressed in SCLC patient samples and was inversely associated with overall survival of the patients. SMARCAL1 was required for SCLC cell proliferation and genome integrity. Mass spectrometry revealed that PAR6B was a downstream SMARCAL1 signal molecule which rescued inhibitory effects caused by silencing of SMARCAL1. By screening of 36 FDA-approved clinically available agents related to DNA damage repair, we found that an aza-anthracenedione, pixantrone, was a potent SMARCAL1 inhibitor which suppressed the expression of SMARCAL1 and PAR6B at protein level. Pixantrone caused DNA damage and exhibited inhibitory effects on SCLC cells in vitro and in a patient-derived xenograft mouse model. These results indicated that SMARCAL1 functions as an oncogene in SCLC, and pixantrone as a SMARCAL1 inhibitor bears therapeutic potentials in this deadly disease.

Application of Epithelial Growth Factor Receptor-Targeted Magnetic Resonance Imaging and Near-Infrared II Dual-Modal Probe in Lung Cancer Diagnosis and Surgical Resection.

There has been an increase in the use of molecular probe diagnostic techniques for lung cancer, and magnetic resonance imaging (MRI) offers specific advantages for diagnosing pulmonary carcinoma. Furthermore, advancements in near-infrared II (NIR-II) fluorescence have provided a new method for precise intraoperative tumor resection. However, few probes combine preoperative diagnosis with intraoperative imaging. This study aims to fill this research void by employing a dual-modal probe that targets the epidermal growth factor receptor for MR and NIR-II imaging, enabling the preoperative diagnosis of lung cancer using MRI and precise intraoperative tumor localization using NIR-II with a single probe. The imaging effects and targeting ability of the probe were confirmed in cell lines, mouse models, and clinical samples. The MR signal decreased within 24 h in the patient-derived xenograft mouse model. The average signal-to-background ratio of NIR-II reached 3.98 ± 0.27. The clinical sample also showed a decrease in the T2 signal using MRI, and the NIR-II optical signal-to-background ratio was 3.29. It is expected that this probe can improve the diagnostic rate of lung cancer using MRI and enable precise intraoperative tumor resection using NIR-II.

SH003 Enhances the Anti-cancer Effects of Dabrafenib on Lung Cancer Harboring BRAF G469A Mutation by Inhibiting the MAPK Signaling Pathway.

BRAF mutations are relatively uncommon in lung cancer. However, the majority of therapies targeting BRAF mutations have been developed exclusively for lung cancer patients with V600E mutations, limiting their effectiveness in treating tumors with the non-V600E BRAF mutations. As a result, there is a need to explore effective therapeutic strategies for patients with lung cancer carrying non-V600 BRAF mutations. Therefore, this study aims to identify a combination treatment approach that effectively targets lung cancer with G469A non-V600 BRAF alteration. The efficacy of drug treatments was assayed using a patient-derived xenograft (PDX) mouse model. Histological analysis was performed using hematoxylin and eosin and immunohistochemical staining. Cell viability and growth were determined using the WST-8 and colony formation assays. Protein levels and apoptosis were analyzed using western blot and flow cytometry, respectively. We demonstrated that the lung cancer cells harboring the non-V600E G469A mutation were responsive to the combination of SH003 and dabrafenib. By utilizing patient-derived xenograft (PDX) models, we identified that this combined treatment induces apoptosis and exhibits antitumor effects through the reduction of ERK signals. The synergistic effect of the combination treatment on BRAF G469A lung cancer cells was consistent with its effects on PDX models, suggesting that the molecular mechanism of apoptosis involves a decrease in the MEK/ERK signaling pathway. The SH003 and dabrafenib combination can be potentially developed as an effective treatment strategy for addressing lung cancer patients with the BRAF G469A mutation.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML

AbstractThe overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor GADD45A is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of LGR4 oncogenic signaling and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition.

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson's disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

Abstract LB178: HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of all breast carcinomas and has poor prognosis. Chemotherapy is the mainstay treatment for TNBC patients, yet with limited clinical benefit. Androgen receptor (AR) is expressed in approximately 50% of TNBC tumors and thus the anti-androgen therapy could be a promising solution for the treatment of AR+ TNBC. Nevertheless, AR inhibitors including enzalutamide and bicalutamide or the androgen biosynthesis inhibitor abiraterone have failed to demonstrate satisfying anti-tumor activity in clinical trials. We have discovered HP518, an AR-targeting PROTAC (proteolysis targeting chimera) that induces potent degradation of both the wild-type AR and AR mutants. HP518 is currently under the clinical development for the treatment of mCRPC. Here we report the preclinical data on HP518 for the treatment of AR+ TNBC. HP518 robustly degrades AR in multiple TNBC cell lines with a half-maximal degradation concentration (DC50) < 1 nM. HP518 strongly inhibits AR+ TNBC cell growth, whereas the AR inhibitor enzalutamide does not show significant anti-proliferative effect on these cells in the presence or absence of androgen. Consistent with the in vitro results, HP518 significantly and dose-dependently inhibits tumor growth in MDA-MB-453 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models. PI3KCA activating mutations are frequently expressed in AR+ TNBC. HP518 in combination with alpelisib, a PI3Kα inhibitor shows a synergistic anti-tumor activity. Mechanistically, HP518, but not AR inhibitor enzalutamide downregulates the mRNA expression levels of a set of DNA replication-related genes in AR+ TNBC cells, indicating a novel and ligand-independent role for AR in regulating growth of AR+ TNBC cells. Therefore, HP518, an AR-targeting PROTAC degrader holds great potential to fulfill the unmet clinical needs for the AR+ TNBC therapy. Selected pre-clinical data will be presented. HP518 structure will not be disclosed. Citation Format: Jing Li, Hongwei Yuan, Kefan Chen, Yongxu Huo, Guoqing Liu, Wu Du, Xinghai Li. HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB178.

Abstract LB177: Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC

Abstract Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men worldwide. Androgens play an important role in the development and progression of prostate cancers and targeting androgen receptor (AR) signaling axis over decades has remained a mainstay of prostate cancer therapy. Next generation hormonal agents (NHAs) enzalutamide and abiraterone have brought clinical benefits for metastatic castrate-resistant prostate cancer (mCRPC) patients, AR axis-targeted therapy resistance is inevitable due to AR gene amplification or mutations. AR-V7 is the most clinically relevant AR splice variant associated with endocrine resistance and poor prognosis. AR-V7 constitutively activates AR signaling and lacks the ligand binding domain (LBD) where hormones and AR antagonists interact, resulting in resistance to AR signaling inhibitors (ARSi). Thus, AR-V7 is a promising therapeutic target to address ARSi resistance in mCRPC. We have discovered a highly potent AR-V7 proteolysis targeting chimera (PROTAC), HC-4955 that degrades both the wild-type AR (WT-AR) and AR-V7 mutant. HC-4955 degrades AR-V7 with DC50 < 5 nM. In silico molecular docking analysis predicts the binding of HC-4955 to the N-terminal domain of AR and then the essential amino acids contributed to HC-4955 binding are validated by mutational analysis studies. HC-4955 also potently degrades mutants of AR-LBD mutations including L702H and T878A and these mutations are also associated with ARSi resistance. HC-4955 strongly inhibits proliferation of ARSi-resistant prostate cancer cell lines with IC50 < 10 nM. HC-4955 shows a robust anti-tumor activity in the AR-V7-bearing 22Rv1 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models, with majority of the tumors completely regressed and > 90% AR-V7 expression decreased at 1-5 mg/kg (QD, PO) in these models. The pre-clinical results support the clinical development of HC-4955 for the treatment of mCRPC. Selected pre-clinical data along with the chemical structure of HC-4955 will be presented. Citation Format: Jing Li, Zhilin Tu, Wu Du, Xi Xiang, Zeyu Wen, Yang You, Lijuan Zhao, Yin Chen, Chengcheng Yang, Hongwei Yuan, Xinghai Li. Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB177.

VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulation

Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness invitro and invivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both invitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.

Abstract 1902: ADRX-0706 Nectin-4 antibody-drug conjugate PK/PD characterization elucidates its widened therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio (DAR) of 8. ADRX-0706 utilizes a novel tubulin inhibitor payload AP052 with a proprietary cleavable linker utilizing Adcentrx’ proprietary i-ConjugationTM technology, which results in excellent biophysical features. Most noteworthy, ADRX-0706 displays a very stable PK profile with overlapping curves for total antibody and ADC as well as full DAR retention in circulation as determined by mass spectrometry. This translates to superior efficacy of ADRX-0706 over EV in multiple in vivo cancer cell line- and patient-derived xenograft mouse models across indications. Detailed PK/PD characterization of ADRX-0706 validates the mechanism of action as an anti-mitotic agent inducing cell cycle arrest and apoptosis. In addition, ADRX-0706 treatment leads to increased PD-L1 expression in mouse pharmacology models, which is a consequence of immunogenic cell death as described for this payload class. These findings are in line with emerging data on EV and provide rationale for combination regimens with checkpoint inhibitors. Besides the improvement in efficacy, ADRX-0706 was also designed for superior safety. A payload distribution study in tumor-bearing mice reveals a preferential and increased delivery of payload to tumors and lower payload delivery to normal tissues as well as lower free payload levels in plasma for ADRX-0706 compared to EV. Taken together, these data provide association of the improved drug-linker stability of ADRX-0706 to an improved Nectin-4 directed delivery of AP052 payload to the tumor and lower exposure in normal tissues, which may translate to an improved therapeutic window. In summary, the preclinical efficacy and safety properties of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Maria Shahmoradgoli, Andrew Hau, Dong Jun Lee, Anna Wang, Peter P. Challita, Oscar Betancourt, Wes Sisson, Mario M. Kuo, Kris Zhang, Albert Goldson, Sam Janssen, Paul Datta, Felipe Acosta, Alexander Chu-Kung, Hui Li, Pia M. Challita-Eid, Sabine Rottmann. ADRX-0706 Nectin-4 antibody-drug conjugate PK/PD characterization elucidates its widened therapeutic window [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 1902.

Abstract 1965: 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors

Abstract FDA-approved pan-FGFR inhibitors bring encouraging results in solid tumors. However, resistance mechanisms and side effect profiles may limit their clinical utility. We designed 3HP-2827, a highly selective and potent small molecule inhibitor of FGFR2, to treat solid tumors harboring FGFR2 alterations, including amplifications, mutations, and fusions to overcome these limitations. 3HP-2827 inhibits FGFR2 enzyme activity with more than 1623-fold selectivity over FGFR1 and FGFR3. 3HP-2827 inhibits cell proliferation with IC50 <10 nM and 30 nM in FGFR2 amplified and FGFR2-mutant cancer cell lines, respectively. 3HP-2827 demonstrates high kinome selectivity for FGFR2 against a panel of 485 human kinases, resulting in 90.1% inhibition of FGFR2, and no other kinases showed greater than 75% inhibition. 3HP-2827 leads only modest activity at 10 µM against PDE4D2 and LCK in 44 distinct common adverse drug reaction targets, supporting its high degree of safety. 3HP-2827 also demonstrates a dose-dependent inhibition of phosphorylation of FGFR2 and FGFR2 signaling pathway nodes, including ERK and AKT, in the FGFR2-dependent cancer cell line. In vivo, 3HP-2827 demonstrates potent antitumor activity in cell- and patient-derived xenograft mouse models, including FGFR2-amplified gastric cancer, FGFR2-mutant (N549K) endometrial cancer, FGFR2 fusion ICC and gastric cancers. Strikingly, 3HP-2827 demonstrates superior activity in patient-derived ICC and gastric cancer, cell-derived endometrial cancer (CDX) in tumor and pFGFR2 inhibitions. In the ICC PDX model, 3HP-2827 also induces the regression of pemigatinib-resistant tumors. 3HP-2827 is well tolerated in the toxicology studies, and no hyperphosphatemia or tissue mineralization is observed at 150 mg/kg/kg/day in rats and 120 mg/kg/day in dogs. The data support that 3HP-2827, a potential to be a best-in-class selective FGFR2 inhibitor, will be studying clinically at the beginning of 2024. Citation Format: Susan S. Liu-Chen, Jufang Lin, Jingyu Hu, Yanchao Liu, Weibo Zhang, Zhixiong Zhang, Yan Ding, Jinqiu Liu, Kevin Chen, Shaojing Hu. 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors [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 1965.

Abstract 3306: RGT-M001, a first-in-class small molecule mRNA degrader of the oncogenic transcription factor c-Myb, demonstrated remarkable single agent anti-tumor efficacy in cancer patient-derived xenograft model

Abstract Dysregulated transcription factors (TFs) represent a unique class of drug targets that drive aberrant gene expression programs involved in the major hallmarks of cancers. However, developing drug-like small molecules targeting TF proteins for clinical purposes has proven to be extremely challenging. Here, we present a novel and innovative approach to degrade the RNA transcript of the master oncogenic transcription factor C-MYB, thereby preventing its undesirable expression, using potent and orally available small molecules. C-MYB is a transcription factor that regulates differentiation and proliferation programs in normal cells, including hematopoietic, stem and epithelial cells. In numerous cancer types, such as adenoid cystic carcinoma (ACC), leukemia, colorectal cancer, and breast cancer, C-MYB is a well-established oncogenic transcription factor, overactivated via different mechanisms, including chromosomal translocations and gene amplification. Here, we leveraged our integrative RNA-targeting platform to identify and validate actionable cryptic exon target sites in the human C-MYB gene. We identified RGT-M001, a potent small molecule that can selectively induce the inclusion of the cryptic exon into the final C-MYB transcripts, resulting in a robust decrease of C-MYB canonical transcripts and C-MYB protein in cells. In a functional assay, we demonstrated that RGT-M001 has potent cell-killing activity against a large panel of cancer cell lines overexpressing C-MYB (EC50 ~ 20 - 300nM), while sparing normal cells. To confirm RGT-M001's on-target effect, we demonstrated a robust correlation between RGT-M001 cell-killing activity and the knockdown of C-MYB RNA and protein. We further investigated the anti-tumor activity of RGT-M001 in an ACC patient-derived xenograft (PDx) mouse model harboring C-MYB-NFBI fusion. Recurrent or metastatic ACC is a malignant neoplasm of predominantly salivary gland origin for which effective approved therapies are lacking; the best reported ORR being 15.6% for lenvatinib. As a single agent, RGT-M001 reduced in vivo C-MYB transcript levels by >80% at peak drug exposure and induced a remarkable tumor growth inhibition response (~70% TGI) in the ACCX11 PDx mouse model, surpassing the therapeutic benchmark Lenvatinib (40% TGI). Importantly, the RGT-M001 regimen was well tolerated. Finally, we showed that the combination of RGT-M001 with the Notch Inhibitor AL-101 resulted in complete inhibition of tumor growth. In conclusion, these data demonstrate that small molecules targeting RNA are a safe and effective approach to address previously undruggable protein targets. Down-regulation of C-MYB by our RNA-targeting small molecules is an attractive therapeutic strategy to treat ACC and other cancers driven by C-MYB dysregulations. Citation Format: Norman Lu, Patricia Soulard, Kay Li, Heather Sadlish, Chris Yates, Xiubin Gu, Ibrahim Kays, Jae Lee, Sam Hasson, Zhiping Weng, Simon Xi, Travis Wager. RGT-M001, a first-in-class small molecule mRNA degrader of the oncogenic transcription factor c-Myb, demonstrated remarkable single agent anti-tumor efficacy in cancer patient-derived xenograft model [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 3306.

Abstract 3390: Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft of triple negative breast cancer

Abstract Introduction: Eliminating triple-negative breast cancer (TNBC) resistance to neoadjuvant chemotherapy is a critical unmet clinical need. TNBC accounts for 20% of breast cancer and is more aggressive due to increased metastasis, high recurrence rate, and significant resistance to chemotherapy (e.g., Doxorubicin, DOX). Napabucasin (NAPA, a small organic molecule) is recognized to kill cancer stem cells by targeting the transcription factor STAT3 pathway and is currently in clinical trials. Our previous in vitro results showed significant effects with hypoxia-responsive targeted polymersomes compared to control in MDA-MB-231 and Patient-Derived Xenograft (PDX) TNBC cells. Further, we observed increased NRP-1 expression in TNBC patient-derived xenograft (PDX) cells under hypoxic (0.2% oxygen) conditions compared to normoxia (21% oxygen). Hence, we hypothesize that targeted hypoxia-responsive polymersome drug therapy would reduce the tumor growth of the PDX mouse model of triple-negative breast cancer in vivo. Methods: We have established the TNBC PDX mouse model with two major phases, initial propagation (F1-F3) and final study phase (F3-F6), for in vivo study (TNBC tumor samples received from Sanford Broadway Clinic, Fargo, IRB approved). We utilized 3-6 months old female adult NSG mice (Jackson Lab) weighing 18-25 g. PDX tumor-bearing female NSG mice were administered polymersome-encapsulated drug (doxorubicin and other groups) by intravenous injection twice a week for 4 weeks and then observed the animals for an additional week. The percentage of tumor volume growth was calculated on these treatment groups and compared to vehicle. The tumors from all the groups were excised and dissected after the treatment and fixed in 10% formalin for histological evaluation. Tumors from all the groups after treatment were stained using hematoxylin and eosin to analyze the tumor core necrosis, and the average area of necrosis was compared among all the groups. Results: The TNBC PDX mouse model treated with DOX alone and a combination of encapsulated DOX with NAPA showed significant weight loss compared to the control (over 4 weeks). DOX-alone treated mice have shown reduced tumor growth and exhibited a significant difference compared to the control group. Notably, the targeted DOX-encapsulated polymersome demonstrated a much lower tumor volume and displayed a marked difference compared to non-targeted vehicles, free-DOX, and saline. In the initial analysis, we observed a higher necrosis in the tumor of the targeted DOX-encapsulated polymersome compared to the control. Summary and Conclusion: Overall, the hypoxia-responsive targeted polymersomes showed potent antitumor activity in the novel TNBC PDX animal model. With further developments, the targeted polymersomes might have translational potential as drug carriers for treating TNBC. Citation Format: Yogaraj S Ramakrishnan, Shubhashri Ambhore, Connor Edvall, Jiyan Mohammed, Sanku Mallik, Daniel M Tuvin, Venkatachalem Sathish. Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft of triple negative breast cancer [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 3390.

Abstract 146: Development of a novel pediatric patient-derived xenograft model with THPO and NDUFB3 mutations

Abstract Familial myeloproliferative neoplasms (MPN) of childhood are extremely rare and have propensity towards malignant transformation such as leukemia and lymphoma. Due to the rarity of these cases, few cellular and mouse models are available for understanding the tumor biology. Therefore, the molecular mechanisms of malignant transformation remain poorly understood limiting the therapeutic progress to treat these conditions. Here, we report the development of a novel pediatric patient-derived xenograft (PDX) mouse model harboring mutations in the THPO and mitochondrial subunit NDUFB3 genes and characterization of their functions. THPO is a regulator of hematopoiesis and induces trilineage hematopoiesis through HSPC proliferation while NDUFB3 is a subunit of the mitochondrial complex 1. We isolated malignant cells from an enlarged spleen sample and blood sample from a pediatric patient with TPO mediated MPN and established a PDX model (PDX-2333) that recapitulates the patient’s disease. Short tandem repeat analyses confirmed an exact match between the PDX-2333 tumors and patient samples. Exome and Sanger sequencing analyses were performed to identify mutations. Immunohistochemistry staining was used for tumor characterization. Wildtype (WT) and mutant forms of NDUFB3 genes were cloned into expression plasmids with a puromycin selection marker and stably transfected in HEK-293 cells. Stable clones of HEK-293 overexpressing WT and NDUFB3 mutants were tested for growth and sensitivity to anti-cancer therapies. We established a novel PDX model of lymphomagenesis harboring unique mutations in THPO and NDUFB3 genes. The THPO (c.-52C>T) mutation in upstream open reading frame causes loss of inhibition of THPO mRNA expression. The NDUFB3 (c.23 A>G) mutation results in a change of amino acid from glutamine 8 (E8) to glycine 8 (G8) (Ndufb3_E8) and is highly conserved in many vertebrate species. HEK-293 cell lines stably overexpressing WT or NDUFB3 mutants were tested for growth and their sensitivity to ruxolitinib, a Jak2 inhibitor and doxorubicin, an FDA-approved treatment for leukemia and lymphoma. Our data showed that cells overexpressing Ndufb3_E8 have increased proliferation and are highly resistant to ruxolitinib and doxorubicin in comparison to the WT NDUFB3. Developing a novel pediatric PDX model and exome sequencing allow us to identify novel genes involved in malignant transformation leading to lymphoma development. Our data demonstrated that mutations in the THPO (c.-52C>T) and NDUFB3 (c.23 A>G) genes play a key role in the pathogenesis of a lymphoma and in regulating drug sensitivity. Citation Format: Jonathan Shilyansky, Casandro J. Chan, Kristen L. Coleman, Mariah Leidinger, Sharathkumar Bhagavathi, Anjali Sharathkumar, Po Hien Ear. Development of a novel pediatric patient-derived xenograft model with THPO and NDUFB3 mutations [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 146.