Clonal evolution & Humanisation of PDX models

Li-Chin Yao¹, Mingshan Cheng¹, Ken-Edwin Aryee4, Pooja Kumar², Nicole Walsh4, Dale Greiner4, Leonard Shultz³, Edison T. Liu², Michael Brehm⁴, and James G. Keck¹

¹ The Jackson Laboratory, Sacramento, CA 95838, USA

² The Jackson Laboratory, Farmington, CT 06032, USA

³ The Jackson Laboratory, Bar Harbor, ME 04609, USA

⁴ The University of Massachusetts Medical School, Worcester, MA 01605, USA

Introduction

The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. A major avenue of our investigation is to generate murine humanized models containing a more complete human hematopoietic system and robust innate immune cell population. Next generation NSG strains include triple transgenic NSG mice (NSG-SGM3) expressing myelosupportive human cytokines KITLG, CSF2, and IL-3.

Material and Methods

We implanted PS4050 melanoma PDX model into humanized NSG-SGM3 mice and NSG mice engrafted with the same HPCs at 3 months post engraftment. Tumors were dissociated for single cell suspensions and immune cell population was analyzed by multicolor flow cytometry. Effect of anti-PD-1 antibody pembrolizumab (Keytruda) on PS4050 growth was evaluated in hu-NSG-SGM3 mice. We depleted PD-L1 expression in NCI-H460 human lung carcinoma cell line by CRISPR. Effect of Ketyruda on PD-L1 deficient xenograft was compared with the mock cell line.

Results and Discussion

We found that hCD45+ cell infiltration into PS4050 tumor was significantly increased in hu-NSG-SGM3 mice as compared to hu-NSG mice engrafted with the same HPC donor (3.7% vs. 1% of viable cells). The majority of tumor infiltrating cells in hu-NSG-SGM3 mice expressed hCD33 (55% of hCD45+) and the percentage was significantly higher than that in hu-NSG mice (13%). hCD3+ T cell infiltration level was similar between these two strains (~20% of hCD45+). PS4050-bearing hu-NSG-SGM3 mice treated with Keytruda showed a significant reduction in tumor growth and the PD-1 levels in tumor infiltrating T cells were greatly reduced by flow cytometry analysis. The overall hCD45+ cell infiltration and the frequencies of hCD4+T, hCD8+T, and hCD33+myeloid cells in tumors remained similar after treatment. Lastly, we observed that the effect of Keytruda on tumor growth reduction in hu-NSG-SGM3 mice is PD-L1-dependent. Keytruda treatment significantly reduced mock-transfected NCI-H460 cell growth. By comparison, PD-L1 KO NCI-H460 cells grew more slowly than the mock cells and lost the response to Keytruda.

Conclusion

Our results indicate that PDX tumor implanted hu-NSG-SGM3 mice serve as an important platform for understanding the interaction between human immune system and tumor micro-environment and for preclinical immuno-oncology efficacy studies.

Bauke de Boer¹, Maurien G. Pruis¹, Peter Keane², Maria Rosaria Imperato², Marije T. Nijk³, Arjan Diepstra⁴, Peter N. Cockerill², , André B. Mulder³, Constanze Bonifer² and Jan Jacob Schuringa¹

¹ Department of Experimental Hematology, Cancer Research Centre Groningen, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands

² Institute for Cancer and Genomic Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham, United Kingdom

³ Department of Laboratory Medicine, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands

⁴ Department of Pathology and Medical Biology, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands

Intra-tumour heterogeneity caused by clonal evolution is a major problem in cancer treatment stratification. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane (PM) proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth and engraftment behavior, as determined by RNA sequencing, DNaseI hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture and xenograft transplantation in our humanized niche mouse leukemia clinic. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.

Introduction

PDX models have shown promise as accurate models of human cancer and can capture the intra-patient heterogeneity. They are therefore becoming the animal models of choice for the development of cancer therapeutics for human patients. We have used these models to model responses of melanoma to targeted therapy (Einarsdottir et al., Oncotarget 2014; Xue et al Nat Med 2017; Lunavat et al., PNAS 2017), to epigenetic therapy (Muralidharan et al., CDD 2017) and to novel drugs (Gad et al., Nature 2014; Warpman-Berglund et al., Ann Oncol 2016; Einarsdottir et al., CDD in press).

Although these studies strengthen the view about the utility of PDX models to study small molecule cancer therapy, they do not reveal the utility of these models to study cancer immunotherapy. To tackle this, we recently developed an autologous immune-humanized PDX models termed PDXv2.0 (Jespersen et al., Nat Commun 2017). This model relies on the simultaneous growth of tumors and tumor-infiltrating lymphocytes (TILs) from the same patients in PDX models. The data suggest that this novel model can predict responses of patients to adoptive T cell transfer (ACT). However, the utility of the model to predict and model other types of immunotherapies have not yet been analysed.

Material and Methods

The latest and modified methods used will be disclosed in the presentation. The remaining methods can be extracted from our recent publication (Jespersen et al., Nat Commun 2017).

Results and Discussion

By using biopsies from many patients, we are able to capture the variation of responses to cancer immunotherapy. Some patient cases will be elaborated on in more detail as their cancer treatment have been modified in response to data obtained in the study. We will also show early data on pancreas cancer immune-humanized PDX models and show how extended analyses associated with PDX modelling sometimes can help patients receive a correct diagnosis (Bagge et al., JCO-PO, in press).

Conclusion

PDX models will remain useful also in the era of cancer immunotherapy.

J Gómez-Miragaya¹, S Morán¹, L Palomero², R Tonda³, M Palafox¹, V Serra⁴, MA Pujana², M Esteller¹, XS Puente⁵ and E González-Suárez¹

¹ PEBC, IDIBELL, Barcelona, Spain

² ProCURE, IDIBELL, Barcelona, Spain

³ CNAG, Barcelona, Spain

⁴ VHIO, Barcelona, Spain

⁵ University of Oviedo, Oviedo, Spain

Introduction

Taxane-based regimens constitute the most common therapeutic option in patients with triple-negative breast cancer (TNBC). However, primary or acquired chemoresistance are common events and count on being the main cause of death in breast cancer patients. Breast cancer patient-derived xenografts(PDX) have emerged as powerful tools for the study of cancer biology and drug treatment response. Specific methylation patterns have been associated to clinical breast cancer subtypes but methylation remains unstudied in breast cancer PDX. We hypothesize that genetic and epigenetic changes contribute to the acquisition of chemoresistance to docetaxel in TNBC patients.

Material and Methods

Aiming to elucidate docetaxel-associated chemoresistance mechanisms, exome-sequencing, genome-wide DNA methylation and transcriptomic profiling have been performed in preclinical breast cancer patient-derived xenograft (PDX) models of different subtypes and with primary and acquired chemoresistance to docetaxel.

Results and Discussion

We found that DNA methylation patterns from breast cancer PDX are closer to breast cancer clinical samples than breast cancer cell lines (BCCLs) and they maintain subtype specific methylation patterns. Triple negative breast cancer (TNBC) PDX models show very stable methylation patterns accompanying chemoresistance acquisition but some critical genes/pathways were unraveled as differentially methylated.

Most genetic changes in the original human metastasis were maintained after serial passages in mice and long-term docetaxel treatment. We identified a chromosomal amplification present in the human metastatic sample and chemoresistant PDX of a BRCA1-mutant, but not in the docetaxel-sensitive PDX, which was further validated in another mutant model. Increased gene expression of multiple genes contained in that region was observed in residual disease and docetaxel resistant tumors of additional TNBC PDX. Clinical data confirmed that this amplification is associated with a subset of TNBC breast cancer patients, a BRCA1 mutational signature and poor survival after chemotherapy.

Conclusion

Our findings reveal tumor clonal dynamics during engraftment and drug treatments and identify differentially methylated genes and a specific amplification which may contribute to the acquisition of resistance to deocetaxel in TNBC.