Patient-derived Xenograft Mouse Models Research Articles

Fratricide-Resistant Anti-CD2 Chimeric Antigen Receptor T-Cells with Endogenous CD2 Knockout Are Highly Effective Against T-Cell Neoplasms

T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms. In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction ( PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion. Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses. We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) ( PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter. In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.

Dual Targeting of ROR1 and BTK Augments the Anti-Lymphoma Activity in Mantle Cell Lymphoma

Background Mantle cell lymphoma (MCL) is a rare and aggressive subtype of non-Hodgkin lymphoma. Development of BTK inhibitors (BTKis) and CAR-T cells revolutionized the treatment paradigm for the patients with MCL. However, this aggressive B-cell lymphoma frequently relapses leading to poor survival. Therefore, novel therapies are in urgent need to overcome the therapeutic resistance. ROR1 is normally expressed during embryonic development but is usually absent or expressed at low levels in adult tissues. However, re-expression of ROR1 was found in various types of cancer, especially in cancer stem/tumor-initiating cells. ROR1 persistently activates oncogenic signaling pathways in the BCR/BTK-independent manner. Accordingly, we hypothesize that dual targeting of ROR1 and BTK may augment the therapeutic efficacy, and thus combination of monoclonal antibody zilovertamab (cirmtuzumab) targeting ROR1, and BTKis will overcome the therapeutic resistance and effectively kill the relapsed/refractory MCL cells. Methods A panel of MCL cell lines and primary patient samples were used in this study. ROR1 expression levels in MCL cell lines and primary MCL samples were determined by flow cytometry and western blotting. For establishment of patient-derived organoid (PDO), the patient samples or MCL cell from PDX tumors were resuspended in culture medium containing cytokine cocktails. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. The established PDOs were applied for drug sensitivity assays. In addition, a patient-derived xenograft (PDX) mouse model was also used for evaluating single agent and combination efficacy of BTKis and zilovertamab (Zilo). Cell viability assays were performed using CellTiter routine or 3D cell viability assay kit (Promega). Results We hypothesize dual targeting of both BTK and ROR1 signaling pathways will augment cytotoxic effect on the MCL cells including major proliferative and minor ROR1 +CD117 + population, whose survival is not dependent on BTK, in tumor mass. To test the hypothesis, we first analyzed the ROR1 expression in MCL cell lines, primary and PDX MCL cells using flow cytometry and immunoblotting analysis. Our results indicated that in comparison with normal PBMC, ROR1 expression were detected in all examined MCL cell lines including JeKo-1, Mino, Z-138, Granta519, JVM2, SP49, Maver-1 and Rec-1. To test whether p53 status relates to sensitivity of MCL cells to BTKi and Zilo, we examined cytotoxic effect of combination of BTKi and Zilo on MCL cell lines (1) harboring mutated TP53 (T P53 Mut): JeKo-1, JeKo-R, Mino, and Mino-R and (2) harboring wild-type TP53 ( TP53 WT): Z-138 and Maver-1, and PDO as well as PDX tumors derived from biopsies bearing either TP53 WT or TP53 Mut. Treatment with single BTKi at 2-10 µM (Ibrutinib, zanubrutinib, acalabrutinib or pirtobrutinib) significantly induced cytotoxicity in the TP53 MutMCL cells, and the cell death was remarkedly increased when BTKi was combined with Zilo at 25-50 µg/ml. In contrast, in MCL cell lines with TP53 WT, BTKi-Zilo combination did not significantly result in increase of cytotoxic effect compared to single agents. These results suggest that the genetic status of TP53 is important for the augmented cytotoxic effect of BTKi-Zilo combination on MCL cells. To further evaluate potentiated effect of BTKi-Zilo combination, we performed ex vivo viability assay using PDO platform. Typically, PDOs were treated with either single agent or combined regimens. Consistent with the data in MCL cell lines, BTKi-Zilo combination resulted in potentiated cytotoxic activity in the PDO models derived from naïve and relapsed patients harboring TP53 Mut. Furthermore, BTKi-Zilo combination displayed synergistic effect in a PDX model derived from a patient harboring TP53 Mut. Conclusion Dual targeting of BTK and ROR1 signaling pathways augmented efficacy selectively in preclinical MCL models with TP53 Mut. These data provide insights to develop tailored therapeutics to improve patient outcome for patients with TP53 mutation.

Acquired Venetoclax Resistance in an In Vivo Model of B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Altered Functions of Apoptosis Regulators

Deregulation of cell death pathways such as apoptosis is a hallmark of cancer and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death signaling. Inhibition of anti-apoptotic molecules of BCL-2 family cell death inhibitors has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to BCL-2 resulting in apoptosis induction and has shown clinical activity in hematologic malignancies, but primary and secondary resistance have also been described. In this study, we aimed to identify resistance mechanisms acquired during VEN treatment leading to therapy failure of BCL-2 inhibition. In order to mimic a clinical situation with consecutive treatment cycles, we employed a patient-derived human BCP-ALL xenograft mouse model with subsequent courses of VEN administration to ALL bearing recipients in vivo. First, we estimated VEN-sensitivities in a series of BCP-ALL PDX samples (N=30) by ex vivo titration of individual VEN-EC 50 values and identified a range of heterogeneous VEN-activities. A VEN-sensitive (low VEN-EC50) PDX-ALL sample with a pro-B ALL phenotype carrying a KMT2A:: MLL1 gene fusion was selected and transplanted onto immunodeficient NOD/SCID recipient mice. Upon leukemia manifestation (presence of 5% human ALL cells in peripheral blood), mice were either treated with VEN (100 mg/kg, two times five days) or vehicle control. Recipients were sacrificed upon onset of leukemia-related morbidity and ALL cells isolated from spleens were transplanted onto secondary recipients, which were again treated with VEN or vehicle. ALL cells isolated from secondary recipients were retransferred onto tertiary mice and again exposed to in vivo VEN-treatment providing a total of three subsequent transplantation and in vivo VEN-therapy cycles. Assessing VEN sensitivities of the ALL cells initially transplanted and isolated after VEN-treatment cycles by using an ex vivo cytochrome c release assay showed increasing VEN-resistance. Accordingly, comparison of VEN to control in vivo treated cells showed significantly decreased sensitivities over treatment cycles. Importantly, also the time periods between treatment and leukemia re-occurrence in mice indicating decreasing VEN-sensitivity became shorter with every additional in vivo therapy course. Paired RNA- seq analysis revealed that transcript levels of the pro-apoptotic molecules BAX and BIM were significantly downregulated in VEN-exposed/resistant ALL as compared to vehicle control treated cells. Basal BH3 profiling analysis showed impaired apoptotic priming (significantly lower cytochrome c release after BIM and PUMA peptide exposure) and decreased dependence on anti-apoptotic BCL-2 and MCL-1 indicating a shift from a pro-apoptotic to a pro-survival state. This shift was also reflected in an e x vivo drug treatment assay showing decreased sensitivity to the MCL-1 inhibitor S63845 and the BCL-XL inhibitor A-1331852 in ALL cells from VEN- treated mice compared to control- treated mice (S63845 EC 50 1.5 vs. 2.2 µM, A-1331852 EC 50 9.3 vs. 15.3 µM). In line with the low BAX levels in ALL cells with acquired VEN-resistance, PDX BCP-ALL samples with intrinsic VEN resistance also showed lower BAX protein expression. Taken together, acquired VEN-resistance was recapitulated in a co-clinical trial model of BCP-ALL with repeated in vivo treatment cycles showing lower drug sensitivities along with increasingly reduced in vivo anti-ALL activity of VEN. Characterization of acquired VEN-resistance revealed decreased functional dependency on anti-apoptotic proteins and downregulation of pro-apoptotic BIM and BAX, thus pointing to an imbalance of pro- and anti-apoptotic molecules, which can be potentially targeted by directed compounds bypassing resistance to specific BCL-2 inhibition.

The Reversible BTK Inhibitor Nemtabrutinib Demonstrates Favorable Antitumor Efficacy and Enhances the Function of CAR T Cells in Mantle Cell Lymphoma

Background Covalent Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the therapy of mantle cell lymphoma (MCL). In January 2023, the reversible BTK inhibitor pirtobrutinib was approved by the FDA for the treatment of relapsed or refractory MCL. Nemtabrutinib is another reversible BTK inhibitor which also targets other kinases. The objective of this study was to evaluate the efficacy of nemtabrutinib alone and in combination with anti-CD19 CAR T cells against MCL. Methods Cell viability was measured following a 72-hour treatment with nemtabrutinib in a panel of MCL cell lines using CellTiter-Glo luminescent cell viability assay (Promega). Annexin V/PI staining was utilized to determine whether nemtabrutinib induces cell death through apoptosis. Patient-derived organoids (PDOs) were generated by culturing patient primary cells in 50% Matrigel (Corning) and cultured in cytokine-containing medium. The PDOs were treated for 72 hours, and viability assays were performed. Western blotting was employed to investigate the drug impact on kinase signaling pathways. Nemtabrutinib treated Mino cells were subjected to bulk RNA sequencing to interrogate the transcriptome profiling. The anti-MCL efficacy of nemtabrutinib was tested in an ibrutinib-resistant patient derived xenograft (PDX) mouse model in vivo. Additionally, the potential synergistic effects of nemtabrutinib and anti-CD19 CAR T cells were examined using the luciferase-expressing MCL cells. Results Nemtabrutinib demonstrated comparable growth inhibitory activity to ibrutinib in MCL cell lines with IC 50 values at micromolar concentrations (IC 50 = 0.7-10.1 μM). Remarkably, in the 3D PDO screening system that mimics the tumor microenvironment for MCL, nemtabrutinib displayed superior anti-MCL efficacy compared to ibrutinib (p < 0.01). The apoptotic potential of nemtabrutinib was further investigated using Annexin V/PI staining, revealing a dose-dependent induction of apoptosis, and demonstrated a similar sensitivity profile across cell lines observed with ibrutinib. Western blotting revealed that nemtabrutinib effectively inhibited phosphorylation at both Tyr223 and Tyr551 sites of BTK, and also dramatically suppressed the activation of Src family kinases, Syk and ERK. Bulk RNA-seq was performed to examine the transcriptome profile following nemtabrutinib treatment. 449 genes were identified to be significantly upregulated and 460 genes were downregulated. Gene set enrichment analysis uncovered a significant decrease in TNFα signaling via NF-κB, inflammatory response, and IFNγ response signalings (FDR < 0.05). In an ibrutinib-resistant PDX mouse model, treatment with nemtabrutinib resulted in a significant reduction in tumor burden and effectively attenuated the tumor involvement in spleen, liver and bone marrow. Furthermore, to enhance efficacy, we investigated the combination of nemtabrutinib with anti-CD19 CAR T cells in luciferase-expressing MCL cells. The result demonstrated that nemtabrutinib enhanced the effector function and anti-MCL activity of anti-CD19 CAR T cells. Conclusion Nemtabrutinib demonstrated favorable anti-MCL efficacy in both in vitro and in vivo studies, the promising synergistic effects observed in combination with CAR T cells warrants further investigation in both preclinical and clinical settings.

KB-Lanra 1001: A Phase 1b/2 Study of Safety, PK, PD, and Preliminary Efficacy of the Selective SYK Inhibitor Lanraplenib in Combination with the FLT3 Inhibitor Gilteritinib in FLT3-Mutated R/R AML

Background and Significance: FMS-like tyrosine kinase (FLT3)-mutation (FLT3 mut) in patients with newly diagnosed and relapsed/refractory (R/R) acute myeloid leukemia (AML) is associated with shorter overall survival (OS) and relapse-free survival. Gilteritinib is an approved therapy for R/R FLT3 mut AML, but reduction in FLT3 mut burden and sustained efficacy are rarely achieved, thus necessitating investigation of rational therapeutic combinations to improve outcomes. Spleen Tyrosine Kinase (SYK) is a non-receptor tyrosine kinase known to regulate intracellular signaling in response to extracellular stimuli via its immunoreceptor tyrosine-based activation motif (ITAM). SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to HOXA9 and MEIS1-facilitated suppression of myeloid progenitor maturation and proliferation. SYK cooperates with internal tandem duplication (ITD)-mutated FLT3 to drive leukemogenesis. Lanraplenib (LANRA) is an oral, potent, selective, next-generation SYK inhibitor with a 24-hour half-life, and once-daily (QD) dosing. In preclinical studies, the combination of LANRA and the FLT3 inhibitor gilteritinib demonstrated strong antileukemic effects ex vivo, resulting in deeper responses, and extended overall survival in FLT3 ITD-driven AML patient-derived xenograft mouse models. LANRA in combination with gilteritinib is currently being evaluated in patients with R/R FLT3 mut AML in the KB-LANRA 1001 trial (NCT05028751). Study Design and Methods: KB-LANRA 1001 is a global, multicenter, open-label phase 1b/2 trial designed to determine the safety and maximally tolerated and/or recommended phase 2 dose (MTD/RP2D) of LANRA in combination with standard dose gilteritinib (120 mg QD) in patients with R/R FLT3 mut AML. Secondary endpoints include evaluating pharmacokinetics (PK), pharmacodynamics (PD), and response evaluation (complete response (CR), CR with partial hematologic recovery (CRh), duration of response (DoR), event-free survival (EFS) and OS of the combination. Key exploratory endpoints include reduction of FLT3 mutvariant allele frequency among patients who achieve CR/CRh, correlation with selected baseline biomarkers that may predict efficacy outcomes, and assessment of LANRA PD properties (including target engagement). Eligibility requirements include documented FLT3 mut (ITD, tyrosine kinase domain [TKD] or both), age ≥18, and receipt of at least 1 prior therapy. Oral LANRA + gilteritinib will be administered daily until progression/relapse, intolerance, or failure to achieve at least a partial response (PR) after 6 cycles. The phase 1b component will follow a 3+3 dose escalation design. A dose-escalation committee (DEC) will decide LANRA dose escalation based on prospectively defined definitions of dose-limiting toxicity and safety metrics. Enrollment of additional patients to dose cohort(s) previously cleared for safety and tolerability by the DEC will be allowed under specific conditions. The phase 2 component will further evaluate safety, PK, PD, and anti-leukemic activity of the combination at the LANRA RP2D

The Combination of Lanraplenib, a Selective SYK Inhibitor, and Gilteritinib, a FLT3 Inhibitor, Targets Aberrant Proliferation and Differentiation Blockade in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive disease characterized by uncontrolled clonal proliferation of poorly differentiated myeloid progenitor cells in the bone marrow and blood. Deregulated spleen tyrosine kinase (SYK) impairs myeloid differentiation and has been implicated in the pathogenesis of AML driven by FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation (FLT3-ITD). While SYK is rarely mutated, it is highly activated in FLT3-ITD-mutated AML and directly phosphorylates FLT3-ITD, resulting in aberrant expression of multiple oncogenic pathways. Lanraplenib (LANRA) is a selective, next-generation, SYK inhibitor that has shown favorable PK, PD, and safety in healthy volunteers and patients with autoimmune diseases. LANRA is currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). By jointly inhibiting the leukemogenic network created by SYK and mutant FLT3, LANRA and gilteritinib potentially provide a new treatment regimen in a patient population with few treatment options. Method: AML cell viability in response to LANRA, gilteritinib or their combination was assessed by CellTiter Glo (CTG) at 5 days. Apoptosis was evaluated by measuring Annexin V and propidium iodide staining by flow cytometry. Myeloid differentiation was assessed by flow cytometry using CD11b and CD14 cell surface markers and/or immunohistochemistry. In addition, RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. In vivo studies were conducted using the MV4;11 cell line-derived xenograft model. LANRA was administered twice daily at a dose of 75 mg/kg via subcutaneous injections, either alone or in combination with gilteritinib at a dose of 2 mg/kg dosed once daily by oral gavage, for 28 days. Results: Treatment of FLT3-ITD mutated AML cells with LANRA results in dose- and time-dependent increase in myeloid differentiation and reductions in leukemic cell viability. RNA-seq analysis of MV4;11 cells revealed that LANRA abrogates multiple downstream FLT3-ITD leukemogenic signaling pathways including JAK/STAT3/5, MAPK, mTOR and OXPHOS. Combined pharmacologic inhibition of SYK with LANRA and FLT3-ITD with gilteritinib results in robust antileukemic effects in preclinical models of FLT3 ITD-driven AML. Additionally, the combination of LANRA and gilteritinib demonstrated increased differentiation and apoptosis induction in vitro. LANRA in combination with gilteritinib showed significant tumor growth inhibition compared to either single agent in a subcutaneous FLT3-ITD AML cell line-derived xenograft mouse model. In line with these observations, a retrospective analysis of bone marrow-engrafted AML cells from a disseminated FLT3-ITD AML patient-derived xenograft mouse model showed evidence of increased myeloid differentiation and inhibition of cell proliferation. Conclusion: In preclinical models, LANRA demonstrates compelling anti-leukemic activity in combination with gilteritinib. By jointly targeting hyperproliferation and differentiation blockade, LANRA and gilteritinib may provide a new therapeutic approach that can be exploited in AML. Additionally, LANRA disrupts downstream signaling of key pathways often associated with resistance to gilteritinib, providing additional mechanistic support for continued testing of LANRA in combination with gilteritinib in FLT3-ITD-mutated AML patients. These studies helped motivate the ongoing clinical combination with gilteritinib in patients with (R/R) FLT3-mutated AML (NCT05028751).

Establishment of a patient-derived xenograft humanized mouse model for hepatoblastoma in children

Objective: To establish a patient-derived xenograft (PDX) humanized mouse model for hepatoblastoma in children. In addition, compare the biological consistency between successfully modeled PDX tumors and primary tumors in children while comparing and analyzing the influence of PDX model modeling success as a key factor. Methods: A PDX tumor model was constructed from fresh tumor tissue samples from 39 children with hepatoblastoma. The tumor growth time and volume size were recorded in detail. Simultaneously, 39 children's data were collected for experimental and clinical analysis. The difference in tumorigenesis rate between different parameters was analyzed by χ (2) test (categorical variable). Continuous variables with a normal distribution were compared using the t-test. Results: After cell passage and pathological diagnosis, 21 cases of hepatoblastoma PDX models were successfully constructed, with a success rate of 53.8% (21/39). Tumor samples from each generation of successfully modeled PDX models had pathology results that were consistent with those of the corresponding primary tumors. The analysis of the key factors affecting the tumor formation rate of PDX revealed that the metastasis rate was more successful in primary tumors than in liver in situ tumors (7/8 vs. 14/31, P = 0.049). However, there was no significant difference between tumor formation rates and pathological subtypes. According to the PDX tumor formation group comparison between the primary tumor and the metastatic tumor, there was no statistically significant difference between the two groups in terms of tumor formation time and tumor volume. Hematoxylin-eosin staining in hepatoblastoma's PDX mouse was consistent with the primary tumor. Immunohistochemistry positivity rates of four proteins, namely hepatocyte antigen (Hepatocyte), phosphatidylinositol glycan 3, β-catenin, and alpha-fetoprotein, in primary tumor tissues and PDX mouse models were 100% vs. 100%, 100% vs. 95.24%, 100% vs. 100%, and 95.24% vs. 85.71%, respectively. Conclusion: A PDX mouse model for hepatoblastoma has been successfully established in children. The tumor formation rate is high, with metastatic tumors having a higher tumor formation rate than primary tumors and transplanted tumors retaining the biological characteristics of primary tumors.

Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its invitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its invivo efficacy. CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, β-tubulin, glypican-3 (GPC3), β-catenin, and c-Myc. Invivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, β-catenin, and c-Myc highlights a novel mechanism of action.

A DNA/RNA heteroduplex oligonucleotide coupling asparagine depletion restricts FGFR2 fusion-driven intrahepatic cholangiocarcinoma

Pemigatinib, a pan-FGFR inhibitor, is approved to treat intrahepatic cholangiocarcinoma (ICC) harboring FGFR2 fusion mutations. Improving its targeting of FGFR2 fusions remains an unmet clinical need due to its pan selectivity and resistance. Here, we report a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting the chimeric site in FGFR2-AHCYL1 (F-A Cho-HDO) that accumulates in ICC through endocytosis of low-density lipoprotein receptor (LDLR), which is highly expressed in both human and murine ICC. F-A Cho-HDO was determined to be a highly specific, sustainable, and well-tolerated agent for inhibiting ICC progression through posttranscriptional suppression of F-A in ICC patient-derived xenograft mouse models. Moreover, we identified an EGFR-orchestrated bypass signaling axis that partially offset the efficacy of F-A Cho-HDO. Mechanistically, EGFR-induced STAT1 upregulation promoted asparagine (Asn) synthesis through direct transcriptional upregulation of asparagine synthetase (ASNS) and dictated cell survival by preventing p53-dependent cell cycle arrest. Asn restriction with ASNase or ASNS inhibitors reduced the intracellular Asn, thereby reactivating p53and sensitizing ICC to F-A Cho-HDO. Our findings highlight the application of genetic engineering therapies in ICC harboring FGFR2 fusions and reveal an axis of adaptation to FGFR2 inhibition that presents a rationale for the clinical evaluation of a strategy combining FGFR2 inhibitors with Asn depletion.

High-fat diet promotes tumor growth in the patient-derived orthotopic xenograft (PDOX) mouse model of ER positive endometrial cancer

Endometrial cancer, one of the common gynecological malignancies, is affected by several influencing factors. This study established a unique patient-derived orthotopic xenograft (PDOX) nude mouse model for the study of influencing factors in ER positive endometrial cancer. The aim of this study was to demonstrate that a high-fat diet can affect the growth of ER positive endometrial cancer PDOX model tumors. The tumor tissues were expanded by subcutaneous transplantation in nude mice, and then the subcutaneous tumor tissues were orthotopically implanted into the nude mouse uterus to establish the PDOX model. After modeling, they were divided into high-fat diet group and normal diet group for 8 weeks of feeding, which showed that high-fat diet significantly promoted tumor growth (P < 0.001) and increased the protein expression level of ERα in tumor tissues. This study demonstrates that PDOX models of endometrial cancer can embody the role of dietary influences on tumor growth and that this model has the potential for preclinical studies of cancer promoting factors.

Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer.

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized invitro. After experimental screening of unique recombinant miRNAs produced invivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies.

High Clinical Concordance of Drug Resistance in Patient-derived Orthotopic Xenograft (PDOX) Mouse Models: First Step to Validated Precise Individualized Cancer Chemotherapy.

Finding an effective drug for individual cancer patients among the many chemotherapies available and ruling out ineffective drugs are important challenges, especially for patients with advanced cancer. To accomplish this goal, we have pioneered and developed the patient-derived orthotopic xenograft (PDOX) nude mouse model for all cancer types, enabling the discovery and evaluation of novel therapeutics, as well as individualized therapy of patients with cancer. PDOX models can more precisely reproduce the original tumor microenvironment (TME) compared to subcutaneous-implanted xenografts including patient-derived xenograft (PDX) models. The present review presents the concordance of drug resistance in individual cancer patients and their PDOX models. There are 28 PDOX publications with 12 PDOX models from patients who were treated with chemotherapy. Sixteen chemotherapeutics were administrated to these patients and all of them were clinically ineffective. In PDOX models established from these patients' tumors, fourteen chemotherapeutics were resistant with a concordance rate of 88%. PDOX models should be established as early as possible from patients to predict and improve outcome. PDOX models mimic the clinical tumor aggressiveness, therefore enabling a high concordance with clinical outcomes. The present review shows a high concordance for drug resistance between cancer patients and their corresponding PDOX models. Future studies will include prospective clinical trials comparing both drug efficacy and resistance in patients and their PDOX models.

The Extract of Pinellia Ternata-Induced Apoptosis of Leukemia Cells by Regulating the Expression of Bax, Bcl-2 and Caspase-3 Protein Expression in Mice

The study aims to lessen the monetary burden on patients and society by decreasing the price of proprietary drugs used in leukemia therapy. Flow cytometry, reverse transcription polymerase chain reaction, western blot, and a patient-derived xenograft mouse model were used to confirm the therapeutic effect of Pinellia ternata extract on leukemia. Three types of leukemia cells (K562, HL-60, and C8166 cell lines) were found to undergo early apoptosis (P ≤ .05) after being exposed to P. ternata extract, as measured by flow cytometry. Reverse transcription polymerase chain reaction results showed that P. ternata extract at both middle (300 μg/mL) and high (500 μg/mL) concentrations was able to down-regulate Bcl-2 and upregulate mRNA expression of Bax and caspase-3. In the patient-derived xenograft mouse model formed by BALB/c-nu/nu nude mice, immunohistochemistry indicated that P. ternata extract effectively suppressed the proliferation of leukemia cells. Therefore, P. ternata extract at 300 μg/mL and 500 μg/mL could effectively inhibit myeloid and lymphocytic leukemia cell proliferation and promote leukemia cell apoptosis by regulating Bax/Bcl-2 and caspase-3.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma

AbstractThe use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell–like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma

Background and AimsThe dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood which hampers the development of novel strategies for eliminating CSCs. MethodsBy small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFβ/Activin-SMAD2/3 signalling pathway. ResultsInhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to Gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. ConclusionsCollectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

LncRNA BCAN-AS1 stabilizes c-Myc via N6-methyladenosine-mediated binding with SNIP1 to promote pancreatic cancer.

C-Myc overexpression contributes to multiple hallmarks of human cancer but directly targeting c-Myc is challenging. Identification of key factors involved in c-Myc dysregulation is of great significance to develop potential indirect targets for c-Myc. Herein, a collection of long non-coding RNAs (lncRNAs) interacted with c-Myc is detected in pancreatic ductal adenocarcinoma (PDAC) cells. Among them, lncRNA BCAN-AS1 is identified as the one with highest c-Myc binding enrichment. BCAN-AS1 was abnormally elevated in PDAC tumors and high BCAN-AS1 level was significantly associated with poor prognosis. Mechanistically, Smad nuclear-interacting protein 1 (SNIP1) was characterized as a new N6-methyladenosine (m6A) mediator binding to BCAN-AS1 via recognizing its m6A modification. m6A-modified BCAN-AS1 acts as a scaffold to facilitate the formation of a ternary complex together with c-Myc and SNIP1, thereby blocking S phase kinase-associated protein 2 (SKP2)-mediated c-Myc ubiquitination and degradation. Biologically, BCAN-AS1 promotes malignant phenotypes of PDAC in vitro and in vivo. Treatment of metastasis xenograft and patient-derived xenograft mouse models with in vivo-optimized antisense oligonucleotide of BCAN-AS1 effectively represses tumor growth and metastasis. These findings shed light on the pro-tumorigenic role of BCAN-AS1 and provide an innovant insight into c-Myc-interacted lncRNA in PDAC.

Nasopharyngeal carcinoma patient-derived xenograft mouse models reveal potential drugs targeting cell cycle, mTOR, and autophagy pathways

BackgroundNasopharyngeal carcinoma (NPC) is associated with Epstein–Barr virus (EBV) infection. To test preclinical NPC drugs, we established two patient-derived xenograft (PDX) mouse models, EBV-positive PDX-B13 and EBV-negative PDX-Li41, for drug screening. MethodsBased on next generation sequencing (NGS) studies, PDX-B13 had CCND1 copy number (CN) gain but CDKN2A CN loss, whereas PDX-Li41 had CDKN2A and RB1 CN loss, TSC1 (negative regulator of mTOR) frameshift deletion mutation, and increased activation of mTOR, a serine/threonine kinase that governs metabolism, autophagy, and apoptosis. Increased mTOR was also associated with poor NPC prognosis. ResultsEverolimus, an mTOR inhibitor, suppressed tumor growth in the two PDX NPC models and had an additive antitumor effect with palbociclib, a CDK4/6 inhibitor. PDX tumors treated with various drugs or untreated were subjected to RNA sequencing, transcriptome profile analysis, and selective Western blotting to understand the interactions between these drugs and gene expression profiles. Palbociclib also suppressed EB viral nuclear antigen (EBNA1) expression in PDX-B13. Everolimus together with autophagy inhibitor, hydroxychloroquine, had additive anti-tumor effect on PDX-B13 tumor. Immunohistochemistry revealed that high mTOR levels were correlated with poor overall survival in patients with metastatic NPC (N = 90). ConclusionsHigh mTOR levels are a poor prognostic factor in NPC, and cell cycle, mTOR and autophagy pathways may serve as therapeutic targets in NPC. In addition, PDX models can be used for efficiently testing potential NPC drugs.

P10.22.B COMBINATION OF ARTS MIMETICS WITH AUTOPHAGY INHIBITORS AS A NOVEL THERAPEUTIC STRATEGY TO OVERCOME APOPTOSIS RESISTANCE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common malignant brain tumour in adults. The current standard of care for GBM patients consists of maximal surgical resection followed by chemotherapy and radiation. Despite such an aggressive treatment approach, the average 5-year survival rate for GBM patients is approximately 5%. One of the main reasons for poor therapy response in the GBM is resistance to programmed cell death, otherwise known as apoptosis. One of the mechanisms behind apoptosis resistance relies on the upregulation of inhibitor of apoptosis proteins (IAPs) which are known to hinder pro-apoptotic proteins and allow cell survival under abnormal conditions such as severe DNA damage. However, there are natural IAP antagonists such as ARTS which are capable of stimulating cell death by promoting the degradation of IAPs. To harness the pro-apoptotic function of IAP antagonists, small peptide mimetics of ARTS have been developed. This project investigates the efficacy of utilizing ARTS mimetics as a novel strategy to overcome apoptosis resistance in the GBM treatment. MATERIAL AND METHODS This study utilizes patient-derived cells with exogenous gain-of-function (GOF) overexpression to introduce full-length ARTS protein as well as pharmacological small peptide mimetics of ARTS to evaluate the response of GBM cells following the promotion of ARTS activity. We also employ a patient-derived orthotopic xenograft mouse model to test novel combinatorial strategies in vivo, where we used a nanoparticle drug delivery system to ensure treatment delivery across the blood-brain barrier (BBB). RESULTS We found that patient-derived GBM cells were highly resistant to ARTS-induced apoptosis. Further molecular investigation unveiled a novel role of ARTS in the upregulation of autophagy as a resistance mechanism. A combination of ARTS GOF or mimetic peptides with autophagy inhibitors mitigated resistance to ARTS-mediated apoptosis and drastically decreased GBM cell proliferation. This combination was further tested in the patient-derived xenograft mouse model, which demonstrated the successful delivery of ARTS mimetic peptides and autophagy inhibitors through the BBB via nanoparticle lipid carriers. Animals treated with a combination of the autophagy inhibitor Spautin-1 and ARTS mimetic peptides demonstrated significantly prolonged survival, confirming the therapeutic potential of such combinatorial approach for GBM treatment. CONCLUSION Altogether, these findings uncover a previously unknown role of ARTS in autophagy regulation and provide evidence that the combination of IAP-antagonist mimetics with autophagy inhibitors can be an effective strategy against highly aggressive GBM brain tumours. This work was supported by the Health Sciences Centre Foundation (HSCF).

198. MODELING CANCER CACHEXIA USING PATIENT TUMOR ORGANOID-DERIVED XENOGRAFTS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Cancer cachexia is a metabolic and functional multifactorial body wasting syndrome and is one of the major causes of death in late-stage patients. Esophageal squamous cell carcinoma (ESCC) presents one of the highest morbidity and mortality rates due to cancer cachexia. However, there is limited effective and approved targeted treatment. Cancer cachexia animal models are highly limited, with few cancer types available as a pre-clinical drug testing platform and with none related to esophageal carcinoma. Methods We have developed a panel of ESCC patient-derived organoid xenograft (PDOX) mouse models based on our latest patient-derived organoid cultures from Hong Kong patients. Results Our PDOX mouse model displays cachexic phenotypes that traditional cell line-derived xenografts lack, including body wasting and functional signs. The slow-proliferating characteristic of the ESCC PDOX on mice allows development of cachexic conditions before the humane endpoint. Our comprehensive panel of PDOX lines allowed us to identify full spectrum and differential cachexia severities across mice bearing different PDOXs, indicating a heterogenous nature and allowing faithful mimicking of various patient conditions. Molecular analysis showed that several classic cachexia-related gene expressions were up-regulated in the isolated liver and inguinal adipose tissues of cachexic mice, along with elevated circulating Il-6 levels. Conclusion Our ESCC PDOX model potentially serves as a useful pre-clinical cachexia-targeted drug testing platform, providing more treatment options for ESCC patients for better disease management. Acknowledgments We acknowledge the Research Grants Council (Theme-based Research Scheme T12–701/17-R to MLL) and the Food and Health Bureau (Health and Medical Research Fund 06171566 to VZY) of the Hong Kong Special Administrative Region for funding support. We acknowledge DSMZ (German Collection of Microorganisms and Cell Culture) for the KYSE cell lines. We acknowledge the University of Hong Kong LKS Faculty of Medicine Centre for PanorOmic Sciences for providing imaging facilities.

Bcl-2 inhibition combined with PPARα activation synergistically targets leukemic stem cell-like cells in acute myeloid leukemia

Persistence of leukemic stem cells (LSCs) is one of the determining factors to acute myeloid leukemia (AML) treatment failure and responsible for the poor prognosis of the disease. Hence, novel therapeutic strategies that target LSCs are crucial for treatment success. We investigated if targeting Bcl-2 and peroxisome proliferator activated receptor α (PPARα), two distinct cell survival regulating mechanisms could eliminate LSCs. This study demonstrate that the Bcl-2 inhibitor venetoclax combined with the PPARα agonist chiglitazar resulted in synergistic killing of LSC-like cell lines and CD34+ primary AML cells while sparing their normal counterparts. Furthermore, the combination regimen significantly suppressed AML progression in patient-derived xenograft (PDX) mouse models. Mechanistically, chiglitazar-mediated PPARα activation inhibited the transcriptional activity of the PIK3AP1 gene promoter and down-regulated the PI3K/Akt signaling pathway and anti-apoptotic Bcl-2 proteins, leading to cell proliferation inhibition and apoptosis induction, which was synergized with venetoclax. These findings suggest that combinatorial Bcl-2 inhibition and PPARα activation selectively eliminates AML cells in vivo and vitro, representing an effective therapy for patients with relapsed and refractory AML.