Mingshan Cheng

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg‐PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient‐ derived xenografts [PDX; non‐small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple‐negative breast cancer (TNBC)] or from a TNBC cell line‐derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune‐engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+T cells, as demonstrated by antibody‐mediated depletion. Thus, tumor‐bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research.—Wang, M., Yao, L.‐C., Cheng, M., Cai, D., Martinek, J., Pan, C.‐X., Shi, W., Ma, A.‐H., De Vere White, R. W., Airhart, S., Liu, E. T., Banchereau, J., Brehm, M. A., Greiner, D. L., Shultz, L. D., Palucka, K., Keck, J. G. Humanized mice in studying efficacy and mechanisms of PD‐1‐targeted cancer immunotherapy. FASEB J. 32,1537‐1549 (2018). www.fasebj.org

The EGFR family members sustain the neoplastic phenotype of ALK+ lung adenocarcinoma via EGR1

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays ‘ab initio’ on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted ‘patient-specific’ therapies.

Design, synthesis, and structure-activity relationships of novel insulin receptor tyrosine kinase activators.

A novel series of symmetrical ureas of [(7-amino(2-naphthyl))sulfonyl]phenylamines were designed, synthesized, and tested for their ability to increase glucose transport in mouse 3T3-L1 adipocytes, a surrogate readout for activation of the insulin receptor (IR) tyrosine kinase (IRTK). A structure-activity relationship was established that indicated glucose transport activity was dependent on the presence of two acidic functionalities, two sulfonamide linkages, and a central urea or 2-imidazolidinone core. Compound 30 was identified as a potent and selective IRTK activator. At low concentrations, 30 was able to increase the tyrosine phosphorylation of the IR stimulated by submaximal insulin. At higher concentrations, 30 was able to increase tyrosine the phosphorylation levels of the IR in the absence of insulin. When administered intraperitoneally (ip) and orally (po), 30 improved glucose tolerance in hypoinsulinemic, streptozotocin-treated rats. These data provide pharmacological validation that small molecule IRTK activators represent a potential new class of antidiabetic agents.

In vitro and in vivo prevention of HIV protease inhibitor‐induced insulin resistance by a novel small molecule insulin receptor activator

Protease inhibitor (PI) therapy for the treatment of patients infected with human immunodeficiency virus is frequently associated with insulin resistance and diabetic complications. These adverse effects of PI treatment result to a large extent from their inhibition of insulin‐stimulated glucose transport. Insulin receptor (IR) activators that enhance the insulin signaling pathway could be effective in treating this resistance. However, there are no agents reported that reverse inhibition of insulin action by PIs. Herein, we describe the effects of TLK19781. This compound is a non‐peptide, small molecule, activator of the IR. We now report in cultured cells, made insulin resistant HIV by PI treatment, that TLK19781 both increased the content of insulin‐stimulated GLUT4 at the plasma membrane, and enhanced insulin‐stimulated glucose transport. In addition, oral administration of TLK19781 with the PI, indinavir improved glucose tolerance in rats made insulin resistant. These results suggest, therefore, that IR activators such as TLK19781 may be useful in treating the insulin resistance associated with PIs.

Antitumor Activity of Osimertinib, an Irreversible Mutant-Selective EGFR Tyrosine Kinase Inhibitor, in NSCLC Harboring EGFR Exon 20 Insertions.

EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non–small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo. The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885–96. ©2018 AACR.

Abstract LB-050: Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy

Mouse models are frequently used to test the therapeutic efficacy of anti-cancer drugs. However, the translation of murine experimental data to treatments for patients with cancer often fails due to significant differences between the species, including the differences in the immune system. Our goal is to bridge this gap and to establish an in vivo preclinical model of human tumor immunotherapy by engrafting immunodeficient mice expressing a partial human immune system with human tumor implants. Humanized NOD-scid IL2Rγ (null) (hu-NSG) mice were initially generated by transplanting NSG mice with human CD34 + hematopoietic stem and progenitor cells (HSPCs) which support human hematopoietic and immune system development. Hu-NSG mice develop functional human T cells and B cells with high levels of TCR excision circles, complex TCR repertoire diversity and antigen-specific T cell proliferative responses. Several types of patient-derived tumors (non small cell lung cancer, sarcoma, triple negative breast cancer and invasive bladder cancer) were successfully implanted into HLA mismatched hu-NSG mice. Tumor growth curves show a delay in tumor growth in hu-NSG compared to non-humanized NSG mice. In a colon cancer xenograft model, treatment with chemotherapy agent (5-FU) or with a therapeutic antibody directed against VEGF (Avastin) resulted in decreased tumor growth. In addition to PDX tumors we have also tested human cancer cell lines. Tumor growth was observed in all hu-NSG mice implanted with human ovarian tumor cell line SKOV3-Luc-D3 cells at different time points post HSPC engraftment, showing no evidence of tumor rejection. Thus, our model of humanized mice bearing tumor-derived xenografts provides opportunities to study both the safety and efficacy of current cancer therapies. Citation Format: Minan Wang, James G. Keck, Mingshan Cheng, Danying Cai, Leonard Shultz, Karolina Palucka, Jacques Banchereau, Carol Bult, Rick Huntress. Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy. [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 LB-050. doi:10.1158/1538-7445.AM2015-LB-050

Modeling of Patient Derived Xenografts in Colorectal Cancer

Developing realistic preclinical models using clinical samples that mirror complex tumor biology and behavior are vital to advancing cancer research. While cell line cultures have been helpful in generating preclinical data, the genetic divergence between these and corresponding primary tumors has limited clinical translation. Conversely, patient-derived xenografts (PDX) in colorectal cancer are highly representative of the genetic and phenotypic heterogeneity in the original tumor. Coupled with high-throughput analyses and bioinformatics, these PDXs represent robust preclinical tools for biomarkers, therapeutic target, and drug discovery. Successful PDX engraftment is hypothesized to be related to a series of anecdotal variables namely, tissue source, cancer stage, tumor grade, acquisition strategy, time to implantation, exposure to prior systemic therapy, and genomic heterogeneity of tumors. Although these factors at large can influence practices and patterns related to xenotransplantation, their relative significance in determining the success of establishing PDXs is uncertain. Accordingly, we systematically examined the predictive ability of these factors in establishing PDXs using 90 colorectal cancer patient specimens that were subcutaneously implanted into immunodeficient mice. Fifty (56%) PDXs were successfully established. Multivariate analyses showed tissue acquisition strategy [surgery 72.0% (95% confidence interval (CI): 58.2–82.6) vs. biopsy 35% (95% CI: 22.1%–50.6%)] to be the key determinant for successful PDX engraftment. These findings contrast with current empiricism in generating PDXs and can serve to simplify or liberalize PDX modeling protocols. Better understanding the relative impact of these factors on efficiency of PDX formation will allow for pervasive integration of these models in care of colorectal cancer patients. Mol Cancer Ther; 16(7); 1435–42. ©2017 AACR.

Abstract LB-C01: Patient-derived tumor xenografts in humanized NSG-SGM3 mice: A new immuno-oncology platform

Humanized mice engrafted with tumors enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently found that humanized NOD-scid IL2Rγnull (NSG) mice bearing patient-derived xenografts (PDX) allow efficacy studies of check-point inhibitors. Next generation NSG strains include triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSG-SGM3). Here we provide a direct comparison of check-point inhibitors evaluation in NSG and NSG-SGM3 mice engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from the same donor and implanted with PDX tumors. Corroborating earlier studies, reconstitution of human immune system in the blood was faster and more robust in NSG-SGM3 compared to NSG recipients throughout the course of the study (18 weeks). Human CD45+ cells reached 25% of total blood cells at week 4 in hu-NSG-SGM3 mice and at week 9 in hu-NSG mice. A majority of blood hCD45+ cells in hu-NSG-SGM3 at week 4 were CD33+ myeloid cells. Circulating hCD3+ T cells reached 10% at week 9 and included regulatory T cells (Tregs), consistent with earlier studies. Hu-NSG mice displayed comparable hCD3+ T cells in the blood only at 12-15 weeks and did not contain Tregs. PDX tumors were then engrafted into partially HLA-matched hu-NSG-SGM3 mice at 9 weeks post engraftment. Two PDX models previously shown to respond to anti-PD1 therapy in hu-NSG mice, BR1126 and LG1306, were used. Treatment with the anti-PD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth in both models. Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies. Citation Format: Li-Chin Yao, Mingshan Cheng, Minan Wang, Jacques Banchereau, Leonard Shultz, Karolina Palucka, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: A new immuno-oncology platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C01.

Abstract IA29: Mice host selection for patient-derived xenograft (PDX) model development and other critical factors for success

The Jackson Laboratory PDX program engages 26 staff members, 5 PIs, and runs thousands of PDX models per year in pre-clinical experiments both for internal scientific experiments and external projects. Over the last 6 years of operation of the PDX program, we have gained a significant amount of experience in all aspects of the process. This experience includes the genomic analysis of the tumors and the establishment of an extensive database annotating many of the 446 PDX models in our US inventory. Herein, we describe some of the characteristics of the system that enhances successful experimentation in this platform. Several factors significantly improve the engraftment rate of tumors1: 1) the degree of immunodeficiency the host mouse - the NOD.Cg-Prkdcscid IL2rgtm1wjl/SzJ (aka, NSG), the NOD.CB17-Prkdcscid/J (aka,NOD-scid), or the NOD.Cg-Rag1tm1momIL2rgtm1wjl/SzJ (aka, NRG) engrafting better than the beige-scid and athymic nude mice; 2) the greater the amount of tissue engrafted, 3) the late or metastatic nature of the tumor, 4) the shorter time from surgery to implantation, 5) the absence of enzymatic dissociation, and 6) orthotopic implantation. Sequencing and expression analysis show the maintenance of the core genomic configuration between PDX and the primary tumors though some genetic differences are noted of indeterminate significance. Human stromal cells, however, tend to be replaced by murine stroma after the first passage. Tumor genetic heterogeneity is maintained through the fourth passage in NSG PDX models as confirmed by deep sequencing of bulk tumor and of individual progenitor clones. Most importantly, however, the tumor responses to systemic therapies appear to reflect the patient response2. Therefore, the use of PDX models to test novel agents may speed pre-clinical drug development. The most important use of PDX systems may be in immuno-oncology given that “humanized” PDX models where implantation of a primary human tumor is implanted in a mouse with a reconstituted human cellular immune system provides a powerful preclinical experimental platform to test new immune modulators in human cancers. We, and others, have shown that humanized NSG mice bearing human tumor xenografts exhibit dramatic responses to immune checkpoint inhibitors that are immune cell and drug dependent3. Immune reconstitution is more complex because of the requirement of human cytokines that are not substituted by their murine counterparts. Engineered mice expressing human cytokines, e.g., IL3, CSF2 (GM-CSF), and KITLG (stem cell factor) in the NSG background (aka, NSG-SGM3 mice), and those expressing CSF1 (M-CSF), IL3, CSF2, and THPO in the C;129S4-Rag2tm1.1Flv IL2rgtm1.1Flv/J background (aka, MITRG mice) after engraftment with human hematopoietic stem cells have been shown to support myeloid cells4 including macrophages absent in standard NSG5. It is anticipated that these “next generation” humanized mice will provide a more nuanced picture of the tumor-immune system interaction6. It is important to understand the challenges and limitations of the PDX platform that can be mitigated to a degree by quality control and study design. There are simple caveats. ~5 % of engrafted solid tumors give rise to EBV positive lymphomas and not the primary tumor. Moreover, ~5-10% of PDX tumors are overgrown by a transformed murine cell especially in late passaged. Thus, stringent histological quality control is necessary which includes the assessment of human cytokeratin, which provides an assessment of murine cancer incursion of solid human cancers. Another concern is that drug dosing for PDX experiments is often very different from that used in human studies. The NSG mice are more sensitive to certain DNA damaging agents and to radiation than NRG or Rag1null mice. Therefore, the structuring of combination studies using genotoxic agents is complicated and should be interpreted with appropriate care. In terms of study design, we have found that each PDX model from an individual patient will give rise to individual tumors in an NSG cohort with significant growth and response variations. Thus, for each treatment arm we have calculated that between 6-8 animals is the minimal number required to attain statistical power of 95-99% to identify efficacy between arms. Moreover, in any treatment arm, it is necessary to assess the response of each individual PDX bearing mouse since a few individual tumors in a cohort may be resistant to a drug whereas the average of the arm shows an overall response. Recently, Gao, et al7 presented an alternative way to conduct PDX studies for drug development where only one mouse per PDX model was used per drug. The overall data (not whether a drug specifically was efficacious in a specific disease type) provided important strategic information in development. They raised an important point and that is that PDX preclinical studies should be structured differently from classical clinical trials to make best use of the platform. Not only the trial design, but even how to call a response should be reexamined. The partial responses seen in PDX experiments that can be precisely quantified but that do not qualify using RECIST criteria provide potentially important information about drug efficacy. Taken together, the PDX platform using severely immunodeficient mice is a powerful tool that can significantly accelerate the development of new therapeutics by dramatically facilitating the advancement of innovative therapies with a high likelihood for success8. References: 1Shultz LD, Goodwin N, Ishikawa F, Hosur V, Lyons BL, Greiner DL. Human cancer growth and therapy in immunodeficient mouse models. Cold Spring Harbor Protoc. 2014 Jul 1;2014(7):694-708. 2Garralda E, Paz K, Lopez-Casas PP, Jones S, Katz A, Kann LM, Lopez- Rios F, Sarno F, Al-Shahrour F, Vasquez D, Bruckheimer E, Angiuoli SV, Calles A, Diaz LA, Velculescu VE, Valencia A, Sidransky D, Hidalgo M. Integrated next-generation sequencing and avatar mouse models for personalized cancer treatment. Clin Cancer Res. 2014 May 1;20(9):2476-84 Epub 2014 Mar 14. 3Wang M, Keck JG, Cheng M, Cai D, Shultz L, Palucka K, Banchereau J, Bult C, Huntress R. Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy. [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 LB-050. 4Billerbeck E, Barry WT, Mu K, Dorner M, Rice CM, Ploss A. Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3- expressing NOD-SCID IL2Rγ(null) humanized mice. Blood. 2011 Mar 17;117(11):3076-86. 5Rongvaux A, Willinger T, Martinek J, Strowig T, Gearty SV, Teichmann LL, Saito Y, Marches F, Halene S, Palucka AK, Manz MG, Flavell RA. Development and function of human innate immune cells in a humanized mouse model. Nat Biotechnol. 2014 Apr;32(4):364-72. 6Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol. 2012 Nov;12(11):786-98. 7Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, Zhang C, Schnell C, Yang G, Zhang Y, Balbin OA, Barbe S, Cai H, Casey F, Chatterjee S, Chiang DY, Chuai S, Cogan SM, Collins SD, Dammassa E, Ebel N, Embry M, Green J, Kauffmann A, Kowal C, Leary RJ, Lehar J, Liang Y, Loo A, Lorenzana E, Robert McDonald E 3rd, McLaughlin ME, Merkin J, Meyer R, Naylor TL, Patawaran M, Reddy A, Roelli C, Ruddy DA, Salangsang F, Santacroce F, Singh AP, Tang Y, Tinetto W, Tobler S, Velazquez R, Venkatesan K, Von Arx F, Wang HQ, Wang Z, Wiesmann M, Wyss D, Xu F, Bitter H, Atadja P, Lees E, Hofmann F, Li E, Keen N, Cozens R, Jensen MR, Pryer NK, Williams JA, Sellers WR. High- throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat Med. 2015 Nov;21(11):1318-25. 8Gandara DR, Mack PC, Bult C, Li T, Lara PN Jr, Riess JW, Astrow SH, Gandour-Edwards R, Cooke DT, Yoneda KY, Moore EH, Pan CX, Burich RA, David EA, Keck JG, Airhart S, Goodwin N, de Vere White RW, Liu ET. Bridging tumor genomics to patient outcomes through an integrated patient-derived xenograft platform. Clin Lung Cancer. 2015 May;16(3):165- 72. Citation Format: Edison T. Liu, Carol Bult, Jeff Chuang, Pooja Kumar, Mingshan Cheng, R. Krishna Murthy Karuturi, Vivek Philip, James Keck, Karolina Palucka, Larry Shultz. Mice host selection for patient-derived xenograft (PDX) model development and other critical factors for success. [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 IA29.