Survival In Preclinical Models Research Articles

Restoring Mitochondrial Function and Muscle Satellite Cell Signaling: Remedies against Age-Related Sarcopenia.

Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and ultrastructural changes of muscle fibers in sarcopenia, mitochondria and their quality control pathways play relevant roles. Mononucleated muscle stem cells/satellite cells (MSCs) have been attributed a critical role in muscle repair after an injury. The involvement of mitochondria in supporting MSC-directed muscle repair is unclear. There is evidence that a reduction in mitochondrial biogenesis blunts muscle repair, thus indicating that the delivery of functional mitochondria to injured muscles can be harnessed to limit muscle fibrosis and enhance restoration of muscle function. Injection of autologous respiration-competent mitochondria from uninjured sites to damaged tissue has been shown to reduce infarct size and enhance cell survival in preclinical models of ischemia-reperfusion. Furthermore, the incorporation of donor mitochondria into MSCs enhances lung and cardiac tissue repair. This strategy has also been tested for regeneration purposes in traumatic muscle injuries. Indeed, the systemic delivery of mitochondria promotes muscle regeneration and restores muscle mass and function while reducing fibrosis during recovery after an injury. In this review, we discuss the contribution of altered MSC function to sarcopenia and illustrate the prospect of harnessing mitochondrial delivery and restoration of MSCs as a therapeutic strategy against age-related sarcopenia.

Abstract 3233: EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [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 3233.

Abstract 4127: Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models

Abstract Despite promising results from recent FDA-approved therapies, many advanced melanoma patients develop resistance to both immunotherapy and targeted therapy. A common resistance mechanism to targeted therapy is upregulation of the PI3K/AKT signaling pathway, which has also been shown to promote the development of melanoma brain metastases. Historically, AKT inhibitors have failed in the clinic due to their limited efficacy or intolerable toxicity. Proteomic analysis comparing non-metastatic vs brain metastatic primary tumors in mice revealed focal adhesion kinase (FAK) as an AKT1 specific effector and a potential alternative therapeutic target. FAK is a non-receptor tyrosine kinase that localizes primarily to focal adhesions to regulate cell migration. To determine whether targeting FAK alone or in combination with the RAF/MEK inhibitor avutometinib reduces brain metastases and prolongs survival, we utilized both autochthonous and syngeneic melanoma mouse models. Mice with either subcutaneous tumors or established brain metastases were treated with FAK inhibitor, RAF/MEK inhibitor, or the combination of FAK and RAF/MEK inhibitors. Each cohort was assessed for tumor onset, growth, metastasis, and survival. Our results show that combined RAF/MEK/FAK inhibition significantly delays tumor onset, causes regression of established tumors, prevents the development of brain metastases, promotes the regression of established brain metastases, and prolongs survival. In addition, patient-derived BRAF V600E melanoma xenograft mouse models resistant to the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib, were sensitive to combined RAF/MEK/FAK blockade. The addition of the BRAF inhibitor encorafenib to these models further enhanced the effect on tumor growth. These results support the initiation of a clinical trial evaluating the efficacy of the RAF/MEK inhibitor avutometinib in combination with the FAK inhibitor defactinib in patients with brain metastases from cutaneous melanoma. Additionally, we are assessing non-canonical roles of FAK in modulating the tumor microenvironment to determine whether avutometinib and defactinib also enhance the efficacy of immune checkpoint inhibition in this disease. Citation Format: Karly A. Stanley, Jared D. Almazan, Tursun Turapov, David A. Kircher, Gennie L. Parkman, MiKaela N. Field, Katie M. Culver, Christopher M. Stehn, Silvia Coma, Jonathan A. Pachter, Howard Colman, Sheri L. Holmen. Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models [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 4127.

A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation

BackgroundPhosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models.MethodsKTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC–MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR.ResultsKTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8+ T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models.ConclusionKTC1101's dual mechanism of action—directly inhibiting tumor cell growth and dynamically enhancing the immune response— represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations.

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

VEGF and Other Gene Therapies Improve Flap Survival-A Systematic Review and Meta-Analysis of Preclinical Studies.

Surgical flaps are basic tools in reconstructive surgery. Their use may be limited by ischemia and necrosis. Few therapies address or prevent them. Genetic therapy could improve flap outcomes, but primary studies in this field present conflicting results. This systematic review and meta-analysis aimed to appraise the efficacy of external gene delivery to the flap for its survival in preclinical models. This review was registered with PROSPERO (CRD42022359982). PubMed, Embase, Web of Science, and Scopus were searched to identify studies using animal models reporting flap survival outcomes following any genetic modifications. Random-effects meta-analysis was used to calculate mean differences in flap survival with accompanying 95% CI. The risk of bias was assessed using the SYRCLE tool. Subgroup and sensitivity analyses were performed to ascertain the robustness of primary analyses, and the evidence was assessed using the GRADE approach. The initial search yielded 690 articles; 51 were eventually included, 36 of which with 1576 rats were meta-analyzed. VEGF gene delivery to different flap types significantly improved flap survival area by 15.66% (95% CI 11.80-19.52). Other interventions had smaller or less precise effects: PDGF-13.44% (95% CI 3.53-23.35); VEGF + FGF-8.64% (95% CI 6.94-10.34); HGF-5.61% (95% CI 0.43-10.78); FGF 3.84% (95% CI 1.13-6.55). Despite considerable heterogeneity, moderate risk of bias, and low quality of evidence, the efficacy of VEGF gene therapy remained significant in all sensitivity analyses. Preclinical data indicate that gene therapy is effective for increasing flap survival, but further animal studies are required for successful clinical translation.

ATH-1105, a small-molecule positive modulator of the neurotrophic HGF system, is neuroprotective, preserves neuromotor function, and extends survival in preclinical models of ALS.

Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disorder, primarily affects the motor neurons of the brain and spinal cord. Like other neurodegenerative conditions, ongoing pathological processes such as increased inflammation, excitotoxicity, and protein accumulation contribute to neuronal death. Hepatocyte growth factor (HGF) signaling through the MET receptor promotes pro-survival, anti-apoptotic, and anti-inflammatory effects in multiple cell types, including the neurons and support cells of the nervous system. This pleiotropic system is therefore a potential therapeutic target for treatment of neurodegenerative disorders such as ALS. Here, we test the effects of ATH-1105, a small-molecule positive modulator of the HGF signaling system, in preclinical models of ALS. In vitro, the impact of ATH-1105 on HGF-mediated signaling was assessed via phosphorylation assays for MET, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Neuroprotective effects of ATH-1105 were evaluated in rat primary neuron models including spinal motor neurons, motor neuron-astrocyte cocultures, and motor neuron-human muscle cocultures. The anti-inflammatory effects of ATH-1105 were evaluated in microglia- and macrophage-like cell systems exposed to lipopolysaccharide (LPS). In vivo, the impact of daily oral treatment with ATH-1105 was evaluated in Prp-TDP43A315T hemizygous transgenic ALS mice. In vitro, ATH-1105 augmented phosphorylation of MET, ERK, and AKT. ATH-1105 attenuated glutamate-mediated excitotoxicity in primary motor neurons and motor neuron- astrocyte cocultures, and had protective effects on motor neurons and neuromuscular junctions in motor neuron-muscle cocultures. ATH-1105 mitigated LPS-induced inflammation in microglia- and macrophage-like cell systems. In vivo, ATH-1105 treatment resulted in improved motor and nerve function, sciatic nerve axon and myelin integrity, and survival in ALS mice. Treatment with ATH-1105 also led to reductions in levels of plasma biomarkers of inflammation and neurodegeneration, along with decreased pathological protein accumulation (phospho-TDP-43) in the sciatic nerve. Additionally, both early intervention (treatment initiation at 1 month of age) and delayed intervention (treatment initiation at 2 months of age) with ATH-1105 produced benefits in this preclinical model of ALS. The consistent neuroprotective and anti-inflammatory effects demonstrated by ATH-1105 preclinically provide a compelling rationale for therapeutic interventions that leverage the positive modulation of the HGF pathway as a treatment for ALS.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

A preclinical study to evaluate the combination of azeliragon, an inhibitor of the receptor for advanced glycation endproducts (RAGE), with radiation therapy as a potential treatment of glioblastoma.

e14056 Background: Systemic lymphopenia, which is independently associated with poor overall survival, is observed in approximately 30-50% of patients with glioblastoma (GBM) after chemoradiotherapy. We recently found that cranial irradiation of GBM tumors increases circulating myeloid-derived suppressor cells (MDSC) contributing to systemic lymphopenia. Additionally, we found that cranial radiation increases circulating levels of the RAGE ligands S100A8/9 and HMGB1, which have been reported to increase MDSC and their immunosuppressive effects through binding RAGE. We, therefore, hypothesized that targeting RAGE receptors on MDSC could prevent immunosuppression and improve survival in preclinical GBM models. Methods: We orthotopically implanted GBM cells (GL261 and CT2A) into immunocompetent C57BL6 mice. The GBM tumor-bearing mice were treated with five doses of fractionated radiation therapy (RT, 2 Gy/day for 5 consecutive days) to the whole head in combination with RAGE inhibitor azeliragon (0.1mg/mice/day i.p starting one day before RT for up to 10 days). We evaluated the immune cells in the tumor microenvironment (TME) and spleen at 14 days post-tumor implantation. We also evaluated the circulating immune cells and RAGE ligands (HMGB1 and S100A8/A9) in the plasma at 7, 14, and 21 days post-tumor implantation and monitored the mice for survival up to 60 days. Results: GBM tumor-bearing mice (GL261 & CT2A) treated with RT plus azeliragon had significantly improved survival when compared to RT alone or azeliragon alone. RT plus azeliragon significantly reduced the accumulation of granulocytic-MDSC (G-MDSC), monocytic-MDSC (M-MDSC), tumor-associated macrophages (TAM), and regulatory T cells (Treg), while increasing infiltrating CD8 T cells in the TME as compared to RT alone. RT alone significantly increased the accumulation of G-MDSC and M-MDSC in the spleen, which was significantly reduced with concurrent azeliragon. Concurrent administration of azeliragon with RT did not significantly affect the radiation-induced increase of circulating M-MDSC as well as plasma HMGB1 and S100A8/A9 concentration, though there was a modest reduction of circulating G-MDSC. Conclusions: Our study indicates that azeliragon, by blocking RAGE interaction with its ligands, helps to overcome the MDSC-mediated immunosuppression that occurs after RT. The combination of azeliragon with RT enhances survival in preclinical models. Clinical trials evaluating the safety and efficacy of azeliragon in combination with RT for newly diagnosed GBM are currently under development.

Abstract GS5-07: Estradiol represses anti-tumoral immune response to promote progression of triple-negative breast cancer brain metastases

Abstract Background: Younger women with breast cancer have increased risk of development of brain metastases irrespective of the tumor subtype. We have shown that pre-menopausal levels of 17-β-Estradiol (E2) contributes to the promotion of brain metastases by influencing the tumor microenvironment. E2 promotes brain metastasis (BM) of estrogen receptor negative (ER¯) BC cells by inducing neuroinflammatory ER+ astrocytes in the brain niche to secrete pro-metastatic factors critical for early brain colonization. Ovarieoctomy (OVX) in combination with the aromatase inhibitor (Letrozole) prevented brain colonization of triple negative (TNBC) (ER-PR-HER2-) human xenografts (MDA231BR/NSG) and murine models (E0711/C57Bl6, 4T1/BALBc) through paracrine activation of EGFR and TRKB, pathways involved in increased invasion and early tumor initiation. Yet, the extent to which E2-depletion therapies can decrease progression of established BM in combination with current standard of care for brain metastasis remains unknown. Goal: Current standard of care (SOC) for patients with TNBC brain metastasis includes irradiation (SRS, whole brain) and immunotherapy (PD-1/PDL-1 inhibitors). The goal of this study was to assess how E2-depletion therapies affects brain immune function in the context of SOC for brain metastatic progression of TNBC. Results: To assess whether E2-depletion could decrease BM progression in a model that mimics standard of care for BM, TNBC E0771-GFP-luc cells were injected intracardially in syngeneic ovariectomized (OVX)-female C57Bl6 mice supplemented with pre-menopausal levels of E2. Seven days after injection (when cancer cells have colonized), mice received a single 15Gy dose brain irradiation and were randomized to continue receiving E2, E2 withdrawal (E2WD) or E2WD plus the aromatase-inhibitor letrozole (E2WD+LET). Brain metastatic burden significantly decreased in E2WD and E2WD+Letrozole treated mice as compared to E2-treated mice. Injection of E0711 cells in immunocompromised NSG mice or in the absence of brain irradiation abolished this effect, suggesting that E2-depletion therapies decrease BM progression through boosting radiation-induced anti-tumor immunity. Accordingly, there were no differences in BM progression in E2, E2WD or E2WD+let treated mice in a xenograft model (F2-7 TNBC cells) in NSG mice, even in the presence of brain irradiation. Immune-profiling of brains from OVX+E2, OVX and OVX+Let C57BL6 mice carrying BMs showed dynamic changes in immune populations at early and late stages of brain metastatic colonization. At early stages post brain colonization (3 days post ic injection) E2-treated mice showed a decreased fraction of CD11b+CD45Int CD206+ microglia/CNS macrophages as compared to OVX+LET-treated mice, without significant changes in the fraction of infiltrated lymphocytes, suggesting E2 represses early immunosurveillance through repression of microglia/CNS macrophage activation. At later stages of brain colonization (7 days post ic injection), E2-treated mice showed an increased fraction of proinflammatory microglia and decreased fraction of T and B cells as compared to OVX or OVX+let treated mice. While E2-depletion increased the recruitment of T cells to the brain niche, the fraction of CD279 (PD1+) brain T cells was similar among groups. Ongoing studies assess the efficacy of E2-depletion therapies in combination with brain radiation and PD-1 inhibitors to decrease metastatic burden and improve survival in preclinical models. Conclusion: Our results support the hypothesis that estradiol promotes brain metastatic progression by stimulating an immunosuppressive brain microenvironment. As such, FDA-approved E2-depletion therapies (aromatase inhibitors and selective-estrogen modulators) could be used in combination with brain irradiation and PD-1 inhibitors to promote a more effective anti-tumoral immune response. Citation Format: Diana Cittelly, Maria J. Contreras-Zarate, Karen ALvarez-Eraso, Vesna Tesic, Nicole Tsuji, Leanna Chafee, Sana Karam, D. Ryan Ormond, Peter Kabos. Estradiol represses anti-tumoral immune response to promote progression of triple-negative breast cancer brain metastases [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-07.

Regulation of bone homeostasis by MERTK and TYRO3

The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.

Gene Therapy: Will the Promise of Optimizing Lung Allografts Become Reality?

Lung transplantation is the definitive therapy for patients living with end-stage lung disease. Despite significant progress made in the field, graft survival remains the lowest of all solid organ transplants. Additionally, the lung has among the lowest of organ utilization rates—among eligible donors, only 22% of lungs from multi-organ donors were transplanted in 2019. Novel strategies are needed to rehabilitate marginal organs and improve graft survival. Gene therapy is one promising strategy in optimizing donor allografts. Over-expression or inhibition of specific genes can be achieved to target various pathways of graft injury, including ischemic-reperfusion injuries, humoral or cellular rejection, and chronic lung allograft dysfunction. Experiments in animal models have historically utilized adenovirus-based vectors and the majority of literature in lung transplantation has focused on overexpression of IL-10. Although several strategies were shown to prevent rejection and prolong graft survival in preclinical models, none have led to clinical translation. The past decade has seen a renaissance in the field of gene therapy and two AAV-based in vivo gene therapies are now FDA-approved for clinical use. Concurrently, normothermic ex vivo machine perfusion technology has emerged as an alternative to traditional static cold storage. This preservation method keeps organs physiologically active during storage and thus potentially offers a platform for gene therapy. This review will explore the advantages and disadvantages of various gene therapy modalities, review various candidate genes implicated in various stages of allograft injury and summarize the recent efforts in optimizing donor lungs using gene therapy.

PB1850: A DOSE FINDING AND EXPANSION OPEN LABEL, MULTICENTER STUDY OF IADADEMSTAT AND GILTERITINIB IN PATIENTS WITH RELAPSE/REFRACTORY ACUTE MYELOID LEUKEMIA WITH MUTATED FLT3: THE FRIDA STUDY

Background: Despite improvements in therapy for acute myeloid leukemia (AML), relapses remain a common challenge, contributing to the death of more than 50% of AML patients (pts). Approximately a third of pts with newly diagnosed AML harbor mutations in the fms-like tyrosine kinase 3 (FLT3) gene. Current guidelines recommend molecular testing for FLT3-mut at diagnosis and at relapse, for earlier incorporation of targeted agents FLT3 inhibitors (FLT3i). The approval of the FLT3i midostaurin with induction therapy in FLT3mut AML, and of a more specific potent FLT3i, gilteritinib, as monotherapy for relapsed/refractory (R/R) pts have resulted in improved outcomes. Nevertheless, the duration of remission achieved with single-agent gilteritinib in R/R disease is transient and often brief. Iadademstat (iada) is a highly selective and potent covalent inhibitor of LSD1/KDM1A (lysine demethylase 1) that induces differentiation of AML cells in vitro and decreases leukemic stem cell survival in preclinical models. More than 100 subjects have been treated with iada, including R/R AML pts with monotherapy and naïve AML pts in combination with azacitidine, revealing a manageable safety profile and encouraging response rates. The purpose of the FRIDA clinical trial is to evaluate the safety and effect of iada in combination with the standard-of-care gilteritinib for the treatment of R/R FLT3-mut AML pts. The preclinical rationale supporting the combination is based on: 1) a potent synergistic activity in FLT3-AML in several preclinical models, and 2) the anti-leukemic activity of iada, mediated by preventing the interaction between LSD1 and the GFI1 transcription factor and modifying the epigenetic marks of genes resulting on macrophage/monocytic differentiation. These data, together with iada’s favorable ADME and low DDI risk, warrants further exploration of this combination with the aim of improving outcomes for FLT3-mut R/R AML pts. Aims: The FRIDA study will establish the safety, tolerability and determine the recommended phase 2 dose (RP2D), and preliminary activity for the combination of iada and gilteritinib in pts with FLT3mut R/R AML. Methods: Adult pts with a body weight >50Kg and ECOG 0-2, with relapsed or refractory disease, after 1 or 2 prior lines of therapy, and with FLT3-mutations will be enrolled. In a 3 + 3 escalation phase, up to 18 pts would receive iada at doses of 75 to 150 ug, orally, on 5 days ON -2 days OFF schedule, with continuous gilteritinib PO, at 120 mg/day. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s will be enrolled. Primary endpoints of the study are safety and RP2D determination (based on PK, PD, safety, tolerability, and preliminary activity). Bayesian efficacy monitoring will be performed with cumulating enrollment to ensure futility boundaries are not crossed and to evaluate the activity of the combination. Secondary endpoints include Overall Survival, Event-Free-Survival, Overall Response Rate, Time to Response, Duration of Response and Transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis. Results: This is a clinical trial in progress. No results available. Summary/Conclusion: Study recruiting in Q2 2022 in US.

Abstract 4214: LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation

Abstract Although increased TIGIT expression on TILs is associated with poor survival in patients with cancer, monotherapy TIGIT antibody blockade has not yet demonstrated significant clinical activity. PD-1 is commonly co-expressed on TILs, and PD-1 mediated SHP-2 signaling inhibits DNAM-1 (CD226), preventing PVR co-stimulation in the setting of TIGIT blockade. The interaction between PD-1 and CD226 explains why TIGIT blockade only translates to clinical benefit in the setting of co-blockade of PD-1/L1, however does not explain why co-blockade of TIGIT and PD-1/L1 does not provide benefit in advanced and PD-1/L1 resistant tumors. In advanced and PD-1/L1 resistant tumors, progressive downregulation of CD226 has been reported. Thus, we hypothesized that the lack of CD226 may underlie the lack of clinical responses to combined TIGIT/PD-1/L1 blockade in PD-1/L1 experienced tumors and we sought to identify alternative costimulatory receptors with high-expression. Analysis of TCGA, as well as single-cell RNASeq from TILs, identified HVEM and LTβR as two costimulatory receptors with higher expression in advanced cancers as compared to CD226. HVEM and LTβR directly activate CD8+ T, natural killer (NK), and myeloid cells when bound by their TNF superfamily ligand, known as LIGHT. LIGHT potentiates effector lymphocyte function through signaling that obviates the co-stimulatory role of DNAM-1. We demonstrated that when the extracellular domain (ECD) of LIGHT is combined with the ECD of TIGIT on a TIGIT-Fc-LIGHT bi-functional fusion protein, the simultaneous blockade of PVR, PVRL2, PVRL3, and Nectin-4 and immune co-stimulation by LIGHT increased CD8+ T and NK infiltration into tumors. This translated into tumor cell killing, regression of established tumors, and improved survival in preclinical models of checkpoint primary and acquired resistance. The translation of TIGIT-Fc-LIGHT activity was evaluated in cynomolgus macaques and was well tolerated at doses up to 40 mg/kg. A series of adaptive immune and proinflammatory cytokines, including IL-2, CCL2, CCL4, IL-10, CXCL10, and CCL17, were induced within two hours of TIGIT-Fc-LIGHT infusion. Additionally, receptor engagement led to the rapid margination of HVEM+CD8+ T cells from the periphery into secondary immune tissues. This network of cytokines and post-dose immune cell margination identified in the monkey, was identical to findings in murine models that ultimately translated into significant anti-tumor responses. Lastly, the combination of TIGIT-Fc-LIGHT with anti-PD(L)1 broadened anti-tumor activity of the checkpoint antibodies in aggressive CPI-resistant tumors. These results suggest that LIGHT could be the differentiator that extends the clinical activity of conventional CPIs into PD-L1 low or CPI acquired resistance tumors. Citation Format: Kyung Jin Yoo, Louis Gonzalez, Kellsey Johannes, Jayalakshmi Miriyala, Karen Lenz, Kristen Campbell, Suresh de Silva, Taylor H. Schreiber, George J. Fromm. LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4214.

A phase Ia/Ib study of intrathecal deferoxamine in patients with leptomeningeal metastases.

TPS2074 Background: Leptomeningeal metastases (LM) represent an aggressive form of advanced cancer with few durable therapeutic options. One of the principal barriers in treating LM is the paucity of knowledge on cancer cell survival and proliferation within the nutrient-sparse cerebrospinal fluid (CSF). Single-cell RNA sequencing of patient-derived CSF has identified that cancer cells in the spinal fluid employ the single iron-binding transporter and receptor system, lipocalin-2/SLC22A17, to gather sparse iron to sustain their metabolic needs. This phenotype is recapitulated in preclinical mouse models of LM. Depletion of CSF iron via intracisternal administration of deferoxamine, a parenteral iron chelator, dramatically reduced LM growth and significantly prolonged survival in preclinical models. Exploiting LM iron dependency using intrathecal deferoxamine (IT-DFO) represents a novel therapeutic approach for patients with LM. Methods: This is a prospective, open-label, single center phase Ia dose escalation study of IT-DFO in patients with LM from any solid tumor malignancy to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D), followed by a phase Ib dose expansion of IT-DFO at the RP2D in patients with LM from non-small-cell lung cancer (NSCLC). Eligibility criteria include newly diagnosed or recurrent LM identified by magnetic resonance imaging (MRI), positive CSF cytology, and/or elevated CSF circulating tumor cells (CTCs), age ≥ 18 years, Karnofsky Performance Status ≥ 60, and life expectancy ≥ 8 weeks. All patients will receive IT-DFO in 28-day cycles at a frequency of twice weekly (cycle 1), once weekly (cycle 2), and once every two weeks (cycle 3+). Patients will be monitored for LM progression by neurological examination, neuraxial MRI, and CSF cytology as per modified Response Assessment in Neuro-Oncology LM criteria. Phase Ia will involve a modified accelerated titration over 9 dosing cohorts (IT-DFO dose range 10mg to 495mg) with monitoring for dose-limiting toxicities until the MTD is reached. Phase Ib will further explore the safety of IT-DFO at the RP2D in 20 patients with NSCLC LM. Secondary objectives include the determination of pharmacokinetic and pharmacodynamic properties of IT-DFO and its metabolite, ferrioxamine, in the CSF and serum (phase Ia/Ib), and efficacy outcomes (phase Ib) including LM-objective response rate, LM-clinical benefit rate, LM-duration of response, LM-progression-free survival, and overall survival. Exploratory analyses will prospectively correlate CSF CTC enumeration with treatment response and characterize the impact of IT-DFO on cancer cell metabolism, resistance pathways, and the CSF immune microenvironment. Clinical trial information: NCT05184816.

Combining daratumumab with CD47 blockade prolongs survival in preclinical models of pediatric T-ALL

AbstractAcute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2–like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease–like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.

Transplantation Without Overimmunosuppression (TWO) study protocol: a phase 2b randomised controlled single-centre trial of regulatory T cell therapy to facilitate immunosuppression reduction in living donor kidney transplant recipients

IntroductionRegulatory T cell (Treg) therapy has been demonstrated to facilitate long-term allograft survival in preclinical models of transplantation and may permit reduction of immunosuppression and its associated complications in the...

Combination of tucatinib and neural stem cells secreting anti-HER2 antibody prolongs survival of mice with metastatic brain cancer

Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood-brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation invitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Abstract P256: Pan-ErbB inhibition enhances activity of KRASG12C inhibitors in preclinical models of KRASG12C mutant cancers

Abstract Sotorasib (AMG 510) is the first KRAS inhibitor to be FDA-approved for the treatment of KRASG12C mutant lung adenocarcinomas which comprise ~42% of KRAS mutations in lung adenocarcinomas. Preclinical studies have shown that within hours of KRASG12C inhibition, synthesis of new KRASG12C protein and increased KRAS signaling was observed, leading to enhanced tumor cell growth and survival. This appeared to be due, at least in part, to a feeback loop leading to activation of EGFR signaling but the role of other ErbB family members including ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4) has not been fully evaluated. We hypothesize that short-term adaptation to KRAS inhibition is driven by multiple ErbB family members, and that the combination of pan-ErbB inhibitors with KRASG12C inhibitors could enhance KRASG12C inhibitor activity and prolong survival in preclinical models harboring KRASG12C mutations to a greater extent than more specific EGFR inhibitors. To investigate the role of different ErbB family members in this feedback signaling, we evaluated the impact of an EGFR specific inhibitor, erlotinib, an EGFR/HER2 inhibitor, afatinib, or pan-ErbB inhibitor (EGFR/HER2/3/4), poziotinib on anti-tumor activity of KRASG12C inhibitors in KRASG12C mutant NSCLC cell lines. We initially tested the different inhibitors in combination with sotorasib and adagrasib (MRTX849) and calculated the BLISS index (BI) for each drug pair matrix, using SynergyFinder. We found that when combined with either sotorasib or adagrasib, poziotinib had a synergistic effect (BI>10) in H23 (BI: 17, p=0.01), HCC44 (BI: 11, p=0.04), and H1792 (BI: 13, p=0.01) and an additive effect (BI= -10–10) in H2122 (BI: 2.8). Afatinib and erlotinib were additive in H23 (BI: 9.2 & 9.4), HCC44 (BI:8.8 & 7.9), H2122 (BI: -4.5 & -3.3) and H1792 (BI: 6.6 & 3.3). Poziotinib yielded a higher BI across all four cell lines compared to afatinib (p<0.0001) or erlotinib (p=0.0004). To determine if poziotinib prevented upregulation of ErbB-signaling after treatment with KRASG12C inhibitors, we treated KRASG12C mutant cell lines (NSCLC: H23 and Bladder Cancer: UM-UC-3) with KRASG12C inhibitors, sotorasib or adagrasib, for four hours and found by Western blotting that phosphorylated EGFR, HER2, HER3, and HER4 were increased after treatment. The addition of 10 nM poziotinib prevented the upregulation of phosphorylated EGFR, HER2, HER3, and HER4 in both cell lines. Together these data demonstrate that inhibition of EGFR, HER2, HER3, and HER4 signaling by the pan-ErbB inhibitor poziotinib resulted in greater synergy with KRASG12C inhibitors than EGFR or EGFR/HER2 inhibitors and highlight the potential importance of HER3 and HER4, in addition to EGFR and HER2, in feedback signaling after KRAS G12C inhibition. Additional studies of pan-ErbB and ErbB-specific inhibitors with KRASG12C inhibitors are warranted to determine tolerability and optimal dosing to prolong survival in KRAS mutant cancers. Citation Format: Jacqulyne P. Robichaux, Ana Galan-Cobo, Ried T. Powell, Kelly A. Gale, Jun He, Fahao Zhang, Monique B. Nilsson, Xiang Zhang, Mary M. Sobieski, Nghi Nguyen, Stephan C. Clifford, John V. Heymach. Pan-ErbB inhibition enhances activity of KRASG12C inhibitors in preclinical models of KRASG12C mutant cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P256.

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.