Humanized PDX Models Research Articles

Inhibition of SRC-3 as a potential therapeutic strategy for aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) has a poor prognosis and high relapse rates despite current therapies, necessitating novel treatment regimens. Inhibition of SRC-3 show effectiveness in vivo and in vitro in other B cell lymphomas. Additionally, previous studies have shown that SRC-3 is highly expressed in the lymph nodes of B cell non-Hodgkin's lymphoma patients, suggesting SRC-3 may play a role in the progression of B cell lymphoma. This study aimed to investigate novel SRC-3 inhibitors, SI-10 and SI-12, in mantle cell lymphoma. The cytotoxic effects of SI-10 and SI-12 were evaluated in vitro and demonstrated dose-dependent cytotoxicity in a panel of MCL cell lines. The in vivo efficacy of SI-10 was confirmed in two ibrutinib-resistant models: an immunocompetent disseminated A20 mouse model of B-cell lymphoma and a human PDX model of MCL. Notably, SI-10 treatment also resulted in a significant extension of survival in vivo with low toxicity in both ibrutinib-resistant murine models. We have investigated SI-10 as a novel anti-lymphoma compound via the inhibition of SRC-3 activity. These findings indicate that targeting SRC-3 should be investigated in combination with current clinical therapeutics as a novel strategy to expand the therapeutic index and to improve lymphoma outcomes.

Abstract 4185: Neutralizing human IFN-β combined with anti-human PD-L1 and anti-human CTLA-4 treatment increased control of endometrial tumor growth in a novel HLA-A matched BLT humanized mice

Abstract High concentrations of type I interferon can induce growth arrest and apoptosis in cancer cells upon acute exposure, while low concentrations of the same can provide significant pro-survival benefits upon chronic exposure. In vitro data show the dependence of cancer cells on self-generated IFN-I leads to their demise if the IFN-β gene is disrupted, but they manage to persist when provided with IFN-β from an external source. Importantly, murine models show that inhibition of myeloid-associated INF-β can limit tumor growth. As blockage of type I interferon can also restore the CD8+ T cell function after chronic viral infection, we sought to determine the effects of neutralizing anti-human IFN-β antibody 7859 (N-anti-IFN-β) when added to T-cell directed immunotherapy in BLT humanized mice implanted with HLA-A matched human endometrial cancer PDX specimens. Specifically, we tested the combination of immunotherapy with N-anti-IFN-β combined with anti-human PD-L1 and anti-human CTLA-4. N-anti-IFN-β was first characterized for neutralizing activity to confirm specific neutralization of IFNβ and not human IFN-β. HLA-A matched genotyped CD34 stem cells and thymus (BLT humanized mice) and clinical endometrial tumors (PDX) were joined in BLT humanized mice before infusions with N-anti-IFN-β with or without anti-human PD-L1 and anti-human CTLA-4, anti-human PD-L1 and anti-human CTLA-4 alone, and IgG control antibodies intraperitoneally twice per week over three weeks. Tumor sizes were measured weekly and weighted at the end of the study. Human leukocytes infiltrating PDX were assessed by IHC and flow cytometry. Leukocyte subset activation, exhaustion, and tumor-specific T-cell function were measured. Anti-human PD-L1 and anti-human CTLA-4 reduced tumor growth by 82.9% (p<0.0001), but quickly increased the T cell exhaustion markers PD-1 and LAG3. The addition of N-anti-IFN-β to immunotherapy significantly decreased the tumor growth rate by 96.2% (p<0.0001) also significantly different from immunotherapy alone (p=0.0009) N-anti-IFN-β monotherapy reduced tumor growth rate by 42.4% (p=0.0032). While there was an increase in infiltrating CD4 and CD8 T-cells in combination therapy, reductions in expression of PD-1 and LAG3 were observed when compared to immunotherapy or N-IFN-β groups alone Analysis was done with Prism software. The use of a novel HLA-A matched BLT humanized mice with human tumor PDX model shows that neutralization of IFN-β can increase the T-cell anti-tumor activity and tumor repression of immunotherapy with anti-human PD-L1 and anti-human CTLA-4 treatment. Citation Format: Zhe Yuan, Guorui Zu, Xiao Sun, Emmanouil Papasavvas, Lily Lu, Joel Cassel, Peter L. Lutes, Mark Cadungog, Joseph Salvino, Luis J. Montaner. Neutralizing human IFN-β combined with anti-human PD-L1 and anti-human CTLA-4 treatment increased control of endometrial tumor growth in a novel HLA-A matched BLT humanized mice [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 4185.

Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway.

Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which - when combined with other genetic lesions - result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a-/- HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a-/- LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation-driven myeloid malignancies.

Abstract 5073: NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy

Abstract While the advent of immune checkpoint blockade (ICB) has dramatically improved the prognosis of many immune-infiltrated cancers, for others, unfortunately, these benefits have yet to be realized. The major challenge before the field, then, is to identify combination therapies that act both to combat evolved resistance. NT219 is a novel dual inhibitor of insulin receptor substrates 1 and 2 (IRS) and STAT3. NT219 demonstrated antitumor effects against both in situ and metastatic human melanoma models in mice as a stand-alone treatment and in combination with mutated BRAF and MEK inhibitors. The potential of NT219 to overcome resistance and increase efficacy was demonstrated in PDX models with multiple drug classes. Collectively, these findings provided preclinical proof-of-concept NT219 as a promising novel cancer therapy. Given this data, the goal of our study was to assess the efficacy of combining NT219 with anti-PD-1 and anti-CTLA-4 ICB, and test capacity of the combination to overcome immune resistance. To that end, we have examined PD-L1 expression levels in vitro following NT219 treatment in two different strains of melanoma cells, B16-tdTomato (TMT) and B16 3I-F4, ICB sensitive and ICB resistant derivative lines, respectively. In addition, to evaluate the combination effect of NT219 with ICB therapy in ICB-resistant PDX model, we used a humanized PDX model of pembrolizumab-resistant gastroesophageal tumor (GEJ). These mice were injected with PBMCs from the same patient. Using multiple syngeneic immunocompetent models of melanoma (TMT and 3I-F4 cells) and the humanized PDX model, we investigated the potential of each ICB with NT219. Our findings showed that NT219 increased PD-L1 expression levels on both melanoma cell lines in vitro, however, the PD-L1 expression levels were much more elevated 3I-F4 than TMT cells. Interestingly, the levels of both IRS and activated pSTAT3, the targets of NT219, which consist well known resistance mechanisms, were higher in the resistant cells as compared to the sensitive line. In accordance we found that NT219 was able to restore anti-PD-1 sensitivity in 3I-F4 model (TGI = 58%) in the syngeneic model, promoting tumor rejection and increasing survival rates. The combined approach between NT219 and anti-CTLA-4 displayed a moderate effect (TGI = 37%). In the TMT, NT219 combined with anti-PD-1 showed greater efficacy compared to anti-PD-1 alone (TGI = 63% and TGI = 47%, respectively). These results were confirmed using the humanized ICB-resistant PDX model, where impressive synergy between NT219 and the anti-PD-1 (TGI = 98%) was demonstrated. To summarize, we found a significant synergistic effect of NT219 combined with anti-PD-1 therapy, supported by a mechanism of PDL-1 induction making ICB resistant tumors amenable to ICB treatment. Collectively, these findings demonstrated that NT219 has the potential to reverse ICB resistance in both human PDX and murine syngeneic tumor model systems Citation Format: Ricardo Alexandre de Azevedo, Hadas Reuveni, Menashe Bar-Eli, Michael Curran. NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5073.

Abstract P6-14-05: Regulation of cellular identity and spatial organization during collective breast cancer invasion

Abstract An early step in breast cancer progression is invasion of tumor cells into surrounding tissues. In many breast cancers, particularly ductal carcinomas, this invasion is accomplished by tumor cells migrating as a cohesive group. This often involves cells that take on heterogeneous roles as either leader or follower cells. While studies in common mouse and human breast cancer models have established that leader cells express high levels of keratin-14 (K14) and other basal epithelial markers, the molecular mechanisms regulating K14+ leader cell identity remain obscure. Here we performed time-sampled single cell RNA-sequencing in 3D type I collagen-embedded tumor organoids isolated from the MMTV-PyMT luminal B model of breast cancer. 11 distinct cellular transcriptional states were identified and correlated with K14 expression and invasive strand formation. Having identified the leader cell state we next asked what transcription factors were enriched, reasoning that transcription factors could be master regulators of leader cell fate. 30 different shRNAs targeting 10 genes were systematically evaluated for their effects on collective invasion. From this screen, suppression of Hes1, the downstream target of Notch signaling, yielded a marked switch from collective to single cell invasion. Disseminating single tumor cells maintained high expression of K14 in Hes1 knockdown organoids which was phenocopied by gamma-secretase inhibition in a human TNBC PDX model. Because K14+ tumor cells highly express the Notch ligand Jag1, these results support a model in which Notch signaling, specifically through activation of Hes1, dictates leader cell identity and spatial organization during collective invasion. Studies are ongoing investigating the impact of Hes1 dynamics on leader cell adhesion, hybrid EMT state, and preference for single versus collective metastasis. Because Notch suppression induces leader cell dissemination, we propose that Notch targeted therapy should be combined with therapies eradicating leader cells. Citation Format: Andrea E. Doak, Kevin J. Cheung. Regulation of cellular identity and spatial organization during collective breast cancer invasion. [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 P6-14-05.

ILC3 Expansion in Acute Myeloid Leukemia

Abstract At homeostasis, type 3 innate lymphoid cells (ILC3s) are tissue-resident and have key roles in maintaining mucosal immunity. Recently, the expansion of ILC3s has been described in solid tumors; however, the mechanism driving this finding is not completely understood. In the setting of mouse and human acute myeloid leukemia (AML), we have observed an expansion of ILC3s. Using an AML murine model, we observed a 7-fold increase in ILC3s in the intestine (the normal ILC3 niche) of leukemic mice relative to controls and a 3-fold increase of ILC3s in the bone marrow (intestine: 0.208% +/− 0.04 AML vs 0.032% +/− 0.04 WT, n=5, p<0.001; bone marrow: 1.312% +/− 0.20 AML vs 0.067% +/− 0.20 WT, n=5, p<0.0001). We hypothesized that the ILC3 increase was due at least in part to increased ILC3 differentiation. To address this hypothesis, we isolated the innate lymphoid cell precursor (ILCP) from normal human donors and co-cultured with AML cells which resulted in increased ILC3 frequency (59% +/− 5% with AML vs 28% +/− 5% without AML, n=15, p <0.001). This observation was also recapitulated in a human PDX model in NSG mice (3.22% +/− 7.8% without AML vs 23.91% +/− 7.8% with AML, n=10; p<0.0001). Our published data have shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. By manipulating AHR activity ex vivo, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (60% +/− 5.5% with AML vs 27% +/− 5% AML+AHR inhibitor, n=15, p<0.001). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion by skewing ILCP differentiation towards ILC3. Future studies will define the role of ILC3s in AML progression. Supported by grant from NIH (1R01CA255860-01A1)

Oxphos Inhibition Induces Formation of Tunneling Nanotubes in AML Cells and Facilitates Mitochondrial Transfer from BM Stroma to AML That Contributes to Microenvironment-Mediated Drug-Resistance of AML

Oxphos Inhibition Induces Formation of Tunneling Nanotubes in AML Cells and Facilitates Mitochondrial Transfer from BM Stroma to AML That Contributes to Microenvironment-Mediated Drug-Resistance of AML

P11.65 Insights into the mechanisms of primary brain tumor invasion

Abstract We have made progress in unravelling the mechanisms of tumor cell invasion by focusing the attention on two molecular pathways including chemokines and extracellular matrix molecules. Chemokines are important mediators of cell signaling that operate both on normal cells and tumor cells and in the immune-cell compartment (Billottet et al, 2013). Among the chemokine receptors, CXCR3 mediate diverse biological functions and comes in two major isoforms the A and B isoform. We found that ligand affinities and conformational changes are very different for the A and B form. We have recently elucidated the role and mechanism of CXCR3A in GBM invasion (Boyé et al, 2017b). We demonstrated that agonist stimulation enhances in vitro cell migration and invasion in GBM cells. A major finding was that CXCR3A forms a complex with the trafficking receptor Lipoprotein-related receptor-1 (LRP1). Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to sustained receptor activity and increase in the migration. This was also clinically validated. Our study defines LRP1 as a new regulator of CXCR3 and indicates that targeting CXCR3-A in GBM may constitute a promising strategy to halt tumor cell invasion. The extracellular matrix (ECM) has morphogenic roles in tumors. Important ECM components are the matricellular proteins, called thrombospondins(THBS1-5) (Adams and Lawler 2011). We recently elucidated the complex role of THSB1 in GBM invasion (Daubon et al.2019). Global expression analysis revealed that THBS1 is up-regulated in GBMs and associated with a poor prognosis. We, furthermore, demonstrated that THBS1 did not activate TGFβ in GBM but that TGFβ1 induced the expression of THBS1 via SMAD3. Furthermore, GBM invasion is compromised when THBS1 is silenced in tumor cells. Thus, our data clearly show that THBS1 is not only involved in the regulation of angiogenesis in GBM, but also impacts the invasive behaviour of glioma cells by interacting with a molecule called CD47 expressed on the surface of GBM cells. RNA-sequencing after microdissection of central and peripheral tumour areas in a human PDX model demonstrated that THBS1 was the gene with the highest connectivity in the peripheral invasive tumour areas. Taken together, these data indicate that THBS1 plays important role in the infiltrative process in GBM. REFERENCES: Adams JC, Lawler J. Cold Spring Harb Perspect Biol. 2011;3:a009712 Billottet C, Quemener C, Bikfalvi A. Biochim Biophys Acta. 2013;1836:287- Boyé K et al. Sci Rep. 2017;7:10703 Boyé K et al. Nat Commun. 2017;8:1571 Daubon T et al, Nature Communications. Nat Commun. 2019 Mar 8;10(1):1146 Murphy-Ullrich JE, Poczatek M. Cytokine Growth Factor Rev. 2000 11:59

Evaluating in vivo efficacy \u2013 toxicity profile of TEG001 in humanized mice xenografts against primary human AML disease and healthy hematopoietic cells

Backgroundγ9δ2T cells, which express Vγ9 and Vδ2 chains of the T cell receptor (TCR), mediate cancer immune surveillance by sensing early metabolic changes in malignant leukemic blast and not their healthy hematopoietic stem counterparts via the γ9δ2TCR targeting joined conformational and spatial changes of CD277 at the cell membrane (CD277J). This concept led to the development of next generation CAR-T cells, so-called TEGs: αβT cells Engineered to express a defined γδTCR. The high affinity γ9δ2TCR clone 5 has recently been selected within the TEG format as a clinical candidate (TEG001). However, exploring safety and efficacy against a target, which reflects an early metabolic change in tumor cells, remains challenging given the lack of appropriate tools. Therefore, we tested whether TEG001 is able to eliminate established leukemia in a primary disease model, without harming other parts of the healthy hematopoiesis in vivo.MethodsSeparate sets of NSG mice were respectively injected with primary human acute myeloid leukemia (AML) blasts and cord blood-derived human progenitor cells from healthy donors. These mice were then treated with TEG001 and mock cells. Tumor burden and human cells engraftment were measured in peripheral blood and followed up over time by quantifying for absolute cell number by flow cytometry. Statistical analysis was performed using non-parametric 2-tailed Mann-Whitney t-test.ResultsWe successfully engrafted primary AML blasts and healthy hematopoietic cells after 6–8 weeks. Here we report that metabolic cancer targeting through TEG001 eradicated established primary leukemic blasts in vivo, while healthy hematopoietic compartments derived from human cord-blood remained unharmed in spite of TEGs persistence up to 50 days after infusion. No additional signs of off-target toxicity were observed in any other tissues.ConclusionWithin the limitations of humanized PD-X models, targeting CD277J by TEG001 is safe and efficient. Therefore, we have initiated clinical testing of TEG001 in a phase I first-in-human clinical trial (NTR6541; date of registration 25 July 2017).

Abstract 1702: Assessment of pharmacology and toxicology of anti-CTLA-4 antibody (ADU-1604) in non-human primates and evaluation of local anti-CTLA-4 application

Abstract The immune checkpoint CTLA-4 can be targeted with antibodies that are applied as single agent therapy or in combination with other therapies including surgery, radiation and chemotherapy. Anti-CTLA-4 antibodies may also augment other immunotherapies. Indeed, in syngeneic mouse tumor models anti-CTLA-4 strongly enhanced anti-tumor efficacy of live, attenuated double-deleted Listeria monocytogenes (LADD) and of the STING pathway activator ADU-S100. Anti-CTLA-4 therapy has shown clinical activity and durable responses in advanced cancers, but is also associated with severe immune-related adverse events (irAEs), although manageable and reversible. Local anti-CTLA4 application could limit the induction of irAEs while retaining its capacity to induce and enhance tumor-specific T cell responses. Here we present the results from the preclinical development of ADU-1604, a novel hIgG1 anti-CTLA-4 antibody. ADU-1604 binds a unique epitope on human CTLA-4 and was extensively characterized in vitro demonstrating potent CTLA-4 binding and blockade of CD80 and CD86 and its capability to enhance human T cell responses. As the antibody cross-recognizes CTLA-4 of cynomolgus monkeys this species was selected for the preclinical studies. Male animals received a single intravenous dose of the antibody to assess its pharmacokinetics and pharmacodynamics. ADU-1604 was well tolerated and showed an acceptable half-life. The antibody also enhanced the T cell-dependent antibody response in hepatitis B surface antigen immunized animals. After the single dose PK study a GLP 4-week repeat-dose toxicity study was conducted in cynomolgus monkeys administered 5 doses of ADU-1604 intravenously once a week followed by an 8-week recovery period. In general, ADU-1604 was well tolerated at up to 30 mg/kg/dose. Histopathology data is being processed and recovery data is pending. Proof of concept for the local administration of anti-CTLA-4 antibodies was conducted in syngeneic mouse models with the mouse CTLA-4 antibody 9D9 and in NSCLC humanized PDX models using ADU-1604 in the context of CD45+ human immune cells. These models demonstrated anti-tumor activity of systemically applied ADU-1604, comparable to a reference anti-CTLA-4 antibody. Intratumoral application of ADU-1604 in these models also induced tumor growth inhibition In conclusion, the preclinical development package warrants clinical investigation of ADU-1604 as monotherapy and for future combinations with other Aduro proprietary platforms including STING agonists and (p)LADD. Currently ADU-1604 is progressing through regulatory filing in preparation of clinical development. Citation Format: Maaike Hendriks, Joost Kreijtz, Paul Vink, David Lutje Hulsik, Imke Lodewijks, Astrid Bertens, Jos van de Crommert, Maurice Habraken, Wout Janssen, Judith Stammen-Vogelzang, Olivia Gardner, Weiwen Deng, Meredith Leong, Sarah McWhirter, Hans van Eenennaam, Andrea van Elsas. Assessment of pharmacology and toxicology of anti-CTLA-4 antibody (ADU-1604) in non-human primates and evaluation of local anti-CTLA-4 application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1702.

Abstract A006: Preclinical tumor models in humanized mice for translational immuno-oncology research

Abstract The recent clinical success of immune checkpoint modulators has stimulated immune-oncology research leading to the identification of new tumor immunology targets. However, both target validation and drug development require appropriate humanized preclinical models. Translational research further urgently needs such models for identification of clinically relevant biomarkers and defining rational combination strategies. In previous studies, we have demonstrated that hematopoietic stem cells (HSC) can proliferate and differentiate in immunodeficient mice and establish a functional human immune system with T cells, B cells, NK cells, monocytes, and dendritic cells. Further, we have shown engraftment of patient- (PDX) and cellline-derived xenografts (CDX) on these humanized mice. All CDX and most of the PDX showed no difference in tumor growth compared to non-humanized mice (fully immune resistant). However, some PDX showed a delayed tumor growth on the humanized mice (partly immune resistant), whereas only one model did not grow at all (immune sensitive). This model is demonstrating the functionality of the established human immune system against a PDX. In the next step, we determined PD-L1 expression on different CDX and PDX on RNA and on protein level. Engraftment seems to be dependent on PD-L1 expression on the PDX, with a higher PD-L1 expression correlating with a stronger tumor growth delay. In the last step the PDX/humanized mice system was evaluated for response to the treatment with checkpoint inhibitors alone and in combination therapy with radiation. So far we treated over 20 PDX and CDX models (lung, colon, melanoma, mammary, ovarian, lymphoma) with ipilimumab, nivolumab, and radiation. In most cases we observed a slight tumor growth delay and increased numbers of T cells in the blood and in the tumor. In our fully humanized model system we identified a correlation between response to checkpoint inhibitor treatment and PD-L1 expression on the PDX or CDX. These results demonstrate that fully humanized PDX models enable appropriate preclinical studies on tumor immune biology, evaluation of new immune therapies and combinations, as well as the identification and validation of biomarkers for immune therapy. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Magdalena Paterka, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. Preclinical tumor models in humanized mice for translational immuno-oncology research [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A006.

Abstract B184: TPX-0005, a highly potent TRK inhibitor, effectively overcomes clinical-resistance TRK mutations including solvent front mutants TRKA G595R, TRKB G639R, and TRKC G623R

Abstract The tropomyosin receptor kinases (TRKs), including TRKA/B/C encoded by NTRK1/2/3 genes, are high-affinity receptors for neurotrophins. Oncogenic rearrangement of NTRK1, NTRK2, and NTRK3 have been identified in many solid malignancies. The use of TRK inhibitors entrectinib and larotrectinib has led to clinical benefit in patients with solid malignancies harboring oncogenic NTRK fusions. Similar to ALK and ROS1 inhibitor treatment, the solvent front mutations TRKA G595R and TRKC G623R (both analog to ALK G1202R) were reported in clinic from treatment-resistant patients. A new generation of TRK inhibitors targeting both wild type and mutated TRKs is highly needed for effectively treating patients harboring fusion TRKs. TPX-0005, a novel three-dimensional macrocycle with a much smaller size than current TRK inhibitors in the clinic, was designed to overcome clinical resistance mutations systematically. TPX-0005 potently inhibited both wild type and mutant TRKs including solvent front mutations. TPX-0005 showed pico-molar activity against TRK kinases (IC50s 0.83 nM, 0.05 nM, and 0.10 nM for TRKA/B/C, respectively) in Reaction Biology kinase assay. TPX-0005 is the most potent TRK inhibitor in clinic and effectively overcomes clinical resistance TRK mutations as shown in the table in cell proliferation assays. It was recently reported that LOXO-195 inhibited the phosphorylation of TRKA and ERK kinases in NIH3T3 cell line expressing ΔTRKA G595R or ETV6-TRKC G623R with IC50s of 7 nM and 45.5 nM, respectively. TPX-0005 is more than 10-fold more potent than LOXO-195. In the xenograft tumor model studies including human PDX model, TPX-0005 dramatically caused tumor regression in the tumors carrying WT or solvent-front mutated TRK fusion gene. Overall, TPX-0005 demonstrated desired drug-like properties, good safety profile, and is a supreme TRK inhibitor against WT and mutated TRKs. A phase 1/2 clinical trial of TPX-0005 is actively being pursued (NCT03093116). KM-12 IC50 (nM)Ba/F3 Cell Proliferation Assay IC50 (nM)InhibitorTPM3-TRKAWTLMNA-TRKA WTLMNA-TRKA G595RETV6-TRKB WTETV6-TRKB G639RETV6-TRKC WTETV6-TRKC G623RTPX-0005<0.2<0.20.4<0.20.6<0.23Entrectinib9.20.3705<0.513840.61000Larotrectinib12.33.5102410.9300010.21500 Citation Format: J. Jean Cui, Dayong Zhai, Wei Deng, Evan Rogers, Zhongdong Huang, Jeffrey Whitten, John lim, Yishan Li. TPX-0005, a highly potent TRK inhibitor, effectively overcomes clinical-resistance TRK mutations including solvent front mutants TRKA G595R, TRKB G639R, and TRKC G623R [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B184.

Abstract 1662: Development and validation of humanized mice models implanted with patient derived colorectal cancer xenografts

Abstract Introduction: Agents that reverse T cell inhibitory signals have reinvigorated the strategy of cancer immunotherapy and are leading to robust clinical responses. In colorectal cancer (CRC), objective responses to single-agent therapy with anti-PD-1/PD-L1 antibodies have largely been restricted to the small proportion of CRC patients with microsatellite unstable (MSI-high) disease. Although there are additional subsets of advanced CRC patients that are responsive to immune checkpoint inhibition in combination with tumor-targeted agents, preclinical models have been hampered by their immune-compromised status. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of combination strategies incorporating cancer immunotherapy, we developed a “hematopoietic” humanized mouse model utilizing patient-derived CRC xenografts with the intent of leveraging this model for the development of rational combinations. Methods: BRG.NODSirpalfa newborn pups were humanized through transplantation of 1x105 CD34+ cells purified from umbilical cord blood. Mice were evaluated for chimerism at 8 and 12 weeks. At 16 weeks, tumor tissue from established PDX models was implanted on the right and left flanks of humanized mice. When the average tumor size reached a volume of ~150-300 mm3, the mice were randomized into vehicle or nivolumab (30 mg/kg twice weekly i.p.) treatment groups according to %chimerism. Mice were monitored for signs of toxicity and tumor size was evaluated twice weekly by caliper measurements (tumor volume= (length × width2) × 0.52). At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for immunological assessment. Results: As preliminary proof-of-concept, we successfully established 3 humanized CRC PDX models and one breast cancer cell line (MDA-MB-231). In one of the CRC (MSI-high) and in the breast model we observed tumor growth inhibition in the treated groups vs control. We detected differences in PD-1 expression among treated versus control mice, with lower expression in the nivolumab-treated groups. We also observed an increased number of TILs, CD8+ T cells and greater numbers of T cells in the lymph nodes of treated mice, suggesting T cell expansion. Mice were highly chimeric with high TILs whereas responder tumors exhibited an increase of CD44 high IFN+ T cells, high CD8% and a higher effector memory% (HLADR+, CD45RO+). Conclusions: Humanized PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, Sarah Lindsey Davis, Christopher Lieu, Scott Kopetz, Stacey Bagby, Paul Francoeur, John J. Tentler, Jill Slansky, Roberta Pelanda, Gail Eckhardt. Development and validation of humanized mice models implanted with patient derived colorectal cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2017-1662

Abstract 2668: Efficacy study of immuno-checkpoint antibodies in humanized CDX and PDX models

Abstract Immune therapeutic intervention has been accepted widely for many hematopoietic malignancies and solid malignancies in past several years. Preclinical animal models with reconstitution of immune cells and target-expressing tumor cells were urgently needed for prove of concept studies demonstrating stimulatory immune system activation. Here we describe two established models of human PBMC co-transplantation of CDX and PDX. The study design was based on co-inoculation of human PBMC and PD-L1 expressing cell in NOG mice for reconstitution of immuno-checkpoint blockage. A therapeutic anti-PD1 antibody was applied for immune system activation and tumor growth inhibition measurement. After drugs were administrated i.p. for three weeks, 95 % TGI for A375 CDX model were observed. At the termination of the efficacy study at 28 days post therapeutic intervention, tumors of vehicle (tumor only, PMBC only), PD1 Ab treated groups were collected for immunostaining of CD45. Infiltrating T-cell with CD45+ label was found within the tumors when co-inoculated with human PBMC. PBMC humanized PDX models of ovarian cancer was implanted into mice which reconstituted with human PBMC for therapeutic intervention. Results showed that 85% TGI for PBMC humanized ovarian PDX models was observed. In conclusion, the models established here are feasible for immunotherapeutic evaluation and further additional immune therapeutic test articles can be explored with similar approaches. Citation Format: Feifei Zhang, Yang Yang, Hongkui Chen, Lijun Jia, Kedong Ouyang, Danyi Wen, Taiping Chen. Efficacy study of immuno-checkpoint antibodies in humanized CDX and PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2668. doi:10.1158/1538-7445.AM2017-2668

Abstract IA02: Introduction to the EuroPDX Consortium

Abstract Started in early 2013, EurOPDX gathers 16 academic institutions throughout Europe including 6 Comprehensive Cancer Centres. Each institution is part of a hospital or is collaborating with a neighboring one. EurOPDX includes world-renowned experts at the forefront of research in basic, preclinical, translational and clinical oncology across multiple pathologies, and displays a wide range of expertise in technological platforms. The virtual PDX collection of the consortium consists of more than 1,500 subcutaneous and orthotopic models for more of 30 different pathologies. Our objective is to improve the predictability of preclinical data by (i) improving characterization of the models, (ii) implementing new PDX models to strengthen the representativeness of the collection, and humanized PDX models, (iii) harmonizing working practice, (iv) Integrating complementary models (e.g. ex vivo 2D and 3D assays) in innovative drug development pipelines, and (v) leveraging deep molecular and pharmacogenomic profiling data of the collection of models to discover predictive biomarkers, new targets, and new strategies to overcome drug resistance. In this lecture I will illustrate the value of the consortium with a few examples of collaborative projects involving academic labs but also industrial partners. In particular, I will discuss the current strategies to setting-up the logistics for the performance of multicenter (and sometimes multipathologies) preclinical and co-clinical trials in a collection of fully characterized PDX models. Citation Format: Sergio Roman-Roman. Introduction to the EuroPDX Consortium. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA02.

Abstract 4409: STA-12-8666: a first-in-class HSP90 inhibitor drug conjugate (HDC) designed to selectively deliver chemotherapy to tumors

Abstract Chemotherapeutic drugs have been a mainstay of cancer therapy for decades; however, their effectiveness is often hampered by inefficient drug exposures and undesirable toxicity to normal tissues. Here we report on a novel drug delivery system, termed heat shock protein 90 (HSP90) inhibitor-drug conjugates (HDC), based on the property that small molecule inhibitors of HSP90 are preferentially retained in tumors cells in contrast to their rapid clearance from the circulation and normal tissues. By attaching chemotherapeutic drugs to HSP90 inhibitor backbones, HDC technology exploits this inherent retention property to efficiently deliver cytotoxic payloads directly into tumor tissues and provide extended drug exposure. STA-12-8666 is an HDC that comprises an HSP90 inhibitor fused to the topoisomerase inhibitor SN-38 (active metabolite of irinotecan). In vivo modeling showed that the HSP90 inhibitor moiety was required for tumor-selective retention of STA-12-8666. Prolonged exposure of STA-12-8666 provided extended release of active SN-38 within the tumor compartment, generating up to two weeks of biomarker engagement (γ-H2AX) in contrast to 3-4 days with irinotecan. The broad therapeutic window exhibited by STA-12-8666 conferred superior efficacy and durability over irinotecan treatment alone - resulting in complete or near complete responses (CR) across a broad spectrum of solid tumor models, including an irinotecan-insensitive bladder cancer model and an aggressive lung cancer model where biweekly treatment of STA-12-8666 was initiated at a starting tumor volume 5-times greater that of typical studies. CRs were also observed in a human pancreatic PDX model following 3 doses of STA-12-8666, which were maintained for more than a month. Of note, recurrent PDX tumors remained sensitive to subsequent therapeutic challenge with STA-12-8666 suggesting HDC delivery may circumvent common mechanisms of resistance to irinotecan. Preliminary findings from an ongoing Phase 1 dose escalation study in dogs with spontaneous tumors suggest a well-managed safety profile and encouraging tumor responses. Overall, STA-12-8666 is a promising investigational agent prototypical of a platform technology that can be applied to other cytotoxic payloads to improve therapeutic indices as well as generating new pharmaceutical entities for evaluation as novel anticancer drugs. Citation Format: David A. Proia, Donald L. Smith, Junyi Zhang, Dan Zhou, John-Paul Jimenez, Jim Sang, Sarah Rippy, Cheryl London, Luisa S. Ogawa, Jun Jiang, Teresa Przewloka, Manuel Sequeira, Jaime Acquaviva, Suqin He, John Chu, Chaohua Zhang, Yuan Liu, Josephine Ye, Vladimir Khazak, Igor Astsaturov, Takayo Inoue, Noriaki Tatsuta, Richard C. Bates, Andrew Sonderfan, Dinesh Chimmanamada, Weiwen Ying. STA-12-8666: a first-in-class HSP90 inhibitor drug conjugate (HDC) designed to selectively deliver chemotherapy to tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2015-4409

Abstract 1457: Genomic characterization of a PDX model of T-DM1-resistant HER2+ invasive ductal carcinoma using augmented exome sequencing

Abstract HER2 amplification/overexpression occurs in 20-30% of breast cancers and is associated with poor prognosis and increased metastatic potential. Patients with HER2+ breast cancers who have progressed on trastuzumab and lapatinib are often prescribed the antibody-drug conjugate, do-trastuzumab emtansine (T-DM1). Results from the TH3RESA and EMILIA studies showed that T-DM1 increased progression-free and overall survival compared to standard therapy and suggested it be considered as standard of care. Despite these favorable efficacy results, most patients treated with T-DM1 eventually progress, but the mechanisms of resistance are not understood. Acquired resistance to T-DM1 has been shown in-vitro, but has not been examined in an in-vivo system. An FNA from a metastatic lung lesion was used to establish a human PDX model from a patient with metastatic HER2+ invasive ductal carcinoma. This model was found resistant to T-DM1 administered weekly at 3 m/k and every three weeks at 10 m/k. We performed whole-exome sequencing on both the metastatic tissue and the PDX model using a content-enhanced exome technology we developed. Our augmented exome is optimized to detect major cancer mutations by enhancing coverage over known sequencing gaps and GC-rich regions across >1300 cancer and 200 miRNA genes. We also performed whole-transcriptome sequencing on the PDX model. All data were analyzed using a cancer bioinformatics pipeline optimized for high-accuracy detection of small variants and indels, somatic copy-number aberrations, gene expression and fusions. We comprehensively profiled the exomes of a metastatic HER2+ ductal carcinoma resistant to T-DM1 treatment and a PDX model derived from it. We found that the PDX model was highly consistent with the neoplastic tissue, with respect to both somatic variants and copy-number alterations, establishing it as an accurate representation of a patient-derived T-DM1-resistant tumor. We verified only 0.27% contamination by mouse DNA in the PDX model. Specifically, we confirmed the continued amplification of HER2 as well as CCNE1 and MYC. Loss-of-heterozygosity of TP53 was coupled with a clonal damaging point mutation. Of note, we found a non-synonymous mutation in HER2, suggesting possible involvement in resistance mechanisms. Transcriptome data confirmed mutation expression in the RNA and gene expression changes of amplified/deleted genes. We present the first preclinical model of a human xenograft derived from a HER2+ metastatic breast cancer with acquired T-DM1 resistance. T-DM1 is effective in treating advanced HER2+ breast cancer in patients who have progressed on standard therapies, but this efficacy is short-lived. Here, we used whole-exome and transcriptome sequencing to characterize the genomic profile of tumors that have become resistant to T-DM1 and present a patient-derived model to reveal insights into acquired T-DM1 resistance mechanisms. Citation Format: Elena Helman, Michael J. Wick, Michael J. Clark, Lizette Gamez, Sean Boyle, Kyriakos P. Papadopoulos, Shujun Luo, Anthony W. Tolcher, Parin Sripakdeevong, Mirian Karbelashvili, Deanna Church, Richard Chen, John West. Genomic characterization of a PDX model of T-DM1-resistant HER2+ invasive ductal carcinoma using augmented exome sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2015-1457

Abstract 361: Illuminating the effects of tissue degradation to improve the management of tissues used in cancer research or clinical applications

Abstract The collection and preservation of tissues from surgery to the lab affects the quality and value of research and/or healthcare applications for cancer patients. To this end we are further defining best practices in the management of surgically resected tissues through the analysis of a systems biology model. In the first steps of this process we defined an experimental animal model to assess gene expression signatures of biological pathways related to preservation intervals. Athymic nude murine tissues (kidney, liver, brain, and PDX-models) were excised at baseline and immediately aliquots of tissue were incubated at varying intervals (t=0, 0.5, 1, 3, 6, 9 hours, temperature during intervals was maintained at 37°C). All tissues were preserved at -80C and total RNA was extracted from these tissues using a uniform RNA isolation kit (RNeasy Mini Kit from Qiagen, Inc.) by the same technician. The RNA Integrity Number (RIN) was obtained using the Agilent's BioAnalyzer 2100 and RNA concentration using the Nanodrop 8000. Subsequently, cDNA was synthesized using Thermo Scientific's RevertAid First Strand cDNA Synthesis Kit, and qPCR analysis of each tissue was performed using the Qiagen RT2 Profiler PCR Arrays including the Cell Death PathwayFinder (murine origin liver, kidney, brain) and Hypoxia Signaling Pathway (human origin PDX-model) arrays. Results show a variation of transcripts s that were upregulated (Bax, Bcl2, Fos, Egr1, Tnfrsf10b) or downregulated (Ctss, Hmox1, Epo, Snca) with increased length of tissue incubation and tissue-specific patterns of regulation were observed. Furthermore, these changes in gene expression did not correlate with a decrease in RIN scores, which raises questions about the suitability of RIN as a comprehensive assessment of RNA quality at the transcript level. These findings have important implications for cancer research, namely that tissues that have not been stabilized within several minutes of excision from the host might have undergone degradation of RNA templates that reflect tissue management-related biological activity versus disease-related transcriptome. In future work, we plan to compare macroanalytes such as microRNAs, non-coding RNAs and proteins across different tissue types with an assumption that gene signatures and biological effects will differ per tissue type. We will use results from our experimental biopreservation model to continue the emphasis on transcriptome changes within the context of biological relevance. Citation Format: Scott D. Jewell, Eric Collins, John Beck, David Monsma, Dawna Dylewski, Andrew Borgman, Mary Winn, Galen Hostetter. Illuminating the effects of tissue degradation to improve the management of tissues used in cancer research or clinical applications. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2014-361