Patient-derived Xenograft Mouse Research Articles

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 PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative 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 PO1-29-01.

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.

Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9-dependent AML.

The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP-1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient-derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.

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.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

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.

Abstract LB041: Exploiting a multi-center pre-clinical mouse PDX mini trial to identify new clinical opportunities for protein-drug conjugates (PDCs) targeting ROR1

Abstract The oncofetal cell surface receptor tyrosine kinase ROR1, is expressed on a broad range of solid tumors and hematological malignancies but is largely absent from normal adult tissue. Increased ROR1 expression correlates with poor clinical outcome in certain cancer indications, consistent with its functional role in tumorigenesis, increased metastatic potential and drug resistance. The oncofetal expression pattern of ROR1, coupled with its role in cancer pathology and therapy resistance, and its scaffolding function in caveolae, makes it a highly attractive target for a protein-drug conjugate (PDC) approach. A number of ROR1-targeting ADC and PDC agents are in clinical and pre-clinical development. Understanding the molecular determinants that drive the efficacy of these agents, may open-up patient selection strategies to improve clinical outcomes for ROR1-targeted ADC/PDC therapy. We utilized a small-domain protein-drug-conjugate platform to develop a PDC Candidate Drug (ADP-c389) which produced complete and sustained tumor regressions in patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC) and had an excellent therapeutic index in mice and NHPs. To further inform our clinical planning and assess the broader clinical opportunity afforded by this agent, ADP-c389 was evaluated in a multi-center, multi-indication, 30-PDX mouse mini trial covering 5 different solid tumor indications (lung, ovarian, endometrial, esophageal, gastric). Covariates of target expression, site, model, mouse strain and treatment effect were analysed longitudinally and a multi-metric (TGI, AUC, Best Average Response (BAR), mRECIST) effects analysis undertaken to evaluate response. Efficacy was observed in each of the cancer indications tested, with 77% of models showing an anti-tumor response. Furthermore, the results in NSCLC lung and ovarian PDX models indicated that these are compelling additional cancer types in which to position ADP-c389 clinically. Whilst all responders are ROR1 positive, ROR1 expression itself is necessary but not sufficient to predict the degree of response. Linear Mixed Effect Model (LMM) and multi-omics (RNA-Seq and Proteomics) analysis of the resulting responder/non-responder cohorts has enabled us to determine markers of sensitivity to ADP-c389. In summary, ADP-c389 represents a highly differentiated product for the treatment of a variety of solid tumors. The learnings from this mouse mini trial are also being applied to bi-specific ROR1-based PDCs. Citation Format: Aaron N. Cranston, Mark Wappett, Estelle G. McLean, Chiara Saladino, Aidan McCann, Wei-Wei Kung, Jennifer Thom, Paul Trumper, Andy Porter, Caroline Barelle, Tim Harrison, Graham Cotton. Exploiting a multi-center pre-clinical mouse PDX mini trial to identify new clinical opportunities for protein-drug conjugates (PDCs) targeting ROR1 [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 LB041.

Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis.

Epigenetic reprogramming and escape from terminal differentiation are poorly understood enabling characteristics of liver cancer. Keratin 19 (KRT19), classically known to form the intermediate filament cytoskeleton, is a marker of stemness and worse prognosis in liver cancer. This study aimed to address the functional roles of KRT19 in liver tumorigenesis and to elucidate the underlying mechanisms. Using multiplexed genome editing of hepatocytes in vivo, we demonstrated that KRT19 promoted liver tumorigenesis in mice. Cell fractionation revealed a previously unrecognized nuclear fraction of KRT19. Tandem affinity purification identified histone deacetylase 1 and REST corepressor 1, components of the corepressor of RE-1 silencing transcription factor (CoREST) complex as KRT19-interacting proteins. KRT19 knockout markedly enhanced histone acetylation levels. Mechanistically, KRT19 promotes CoREST complex formation by enhancing histone deacetylase 1 and REST corepressor 1 interaction, thus increasing the deacetylase activity. ChIP-seq revealed hepatocyte-specific genes, such as hepatocyte nuclear factor 4 alpha ( HNF4A ), as direct targets of KRT19-CoREST. In addition, we identified forkhead box P4 as a direct activator of aberrant KRT19 expression in liver cancer. Furthermore, treatment of primary liver tumors and patient-derived xenografts in mice suggest that KRT19 expression has the potential to predict response to histone deacetylase 1 inhibitors especially in combination with lenvatinib. Our data show that nuclear KRT19 acts as a transcriptional corepressor through promoting the deacetylase activity of the CoREST complex, resulting in dedifferentiation of liver cancer. These findings reveal a previously unrecognized function of KRT19 in directly shaping the epigenetic landscape in cancer.

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).

Personalizing non-small cell lung cancer treatment through patient-derived xenograft models: preclinical and clinical factors for consideration.

In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.

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.