The basis of TGCT chemosensitivity and implications for CSC-targeted therapy Our results suggest that the amazingly high survival rates achieved when treating TGCTs with

platinum-based combination chemotherapy are partly due to the presence CSCs with an extremely sensitive damage response. Within this model, the CSCs are depleted upon treatments comparable to the human standard of care and the depletion occurs along with significantly increased survival and reduced tumor burden. Potentially, if tumors were surgically removed from the mice following treatment, as they are in humans, higher murine survival rates may have been achieved.

132

Our data suggest a rapid increase in damage following treatment that quickly induces apoptosis in EC cells (Fig. 2.7, 3.3), but many of the differentiated cells of the teratoma appear less sensitive based on their persistence in vivo and lack of strong apoptotic response in vitro (Fig. 2.6D, 3.2A- D).

These findings are similar to tumors post treatment in humans, which often have surviving teratoma tissue, devoid of EC, when it is surgically removed172,173_{. Additionally, gPAK EC cells}

persist if untreated, and have a relatively high expression of P53 which are both phenotypes of human EC and suggest similar self-renewal and DDR mechanisms30_{. It is important to note that}

seminomas comprise approximately half of all TGCTs but are homogeneous, and do not yet have an identified CSC population. However, they have a slightly better prognosis following genotoxic treatment. One potential explanation for this is that because seminoma cells express many pluripotent transcription factors and have a PGC appearance, most cells may share the same heightened apoptotic response to induced DNA damage as our data shows for EC cells. Additional work is needed to understand the mechanisms of sensitivity in our model and further validate the findings in human PDX models of EC and seminoma.

The discovery of a chemosensitive CSC within a solid cancer that is considered curable is highly relevant to the CSC field. The ultimate goal of efficiently targeting CSCs in otherwise incurable cancers is to render them curable. In our model, we have demonstrated that the standard of care treatment efficiently targets the CSC of TGCTs, which was a devastating and incurable disease before the advent of these drugs. In this context, the efficacy of TGCT treatment suggests that finding ways to eliminate CSCs in somatic cancers may result in similar success stories.

133 4.4 Future directions.

This dissertation work has been focused on the response to therapies known to be efficacious for treating human TGCTs. However, these drugs are toxic to all cells, and while they can cure TGCTs they also cause serious chronic illnesses, such as infertility, which has led to an interest in less toxic treatments162,238_{. Because gPAK murine TGCTs are representative of GCNIS derived human}

TGCTs, this model could provide an ideal system for screening alternative targeted or differentiation therapies. Differentiation therapy is already an attractive alternative for targeting chemoresistant CSCs within a variety of somatic cancers239,240_{. The goal of these therapies is to}

induce differentiation within the CSC population without harming normal stem cell pools. Our data shows that retinoic acid and thioridazine can be effective inducers of differentiation of EC cells in vitro, resulting in loss of tumor-propagating potential (Fig. 3.1A,B). Vitamin D is implicated as a potential differentiation drug for germ cell tumors as well, but the studies rely on long-term cultured EC cells241_{. Although in vitro screening for anticancer drugs is a powerful tool}

in preclinical testing, cells grown for long periods of time show deviations from in vivo tumors, leading to results that are not necessarily biologically relevant (Fig. 4.1). Potential alternative drugs like those mentioned could be tested in vitro in low passage gPAK derived EC cultures before being tested in vivo using the gPAK model mice. Because of this, the gPAK model holds great promise for both understanding the biological mechanisms of these tumors, and for uncovering less toxic candidate drugs for TGCT treatment.

Furthermore, we have made novel and exciting discoveries about the timing of oncogene- induced malignant transformation of germ cells. Our data clearly show that protective mechanisms prevent postnatal oncogenic events in germ cells from inducing tumorigenesis, while also showing evidence that tumorigenesis initiates at E12.5 in our model. However, this timing is only the

134

earliest we can describe for our model, but we can not rule out additional susceptibility between E12.5 and birth. Our results also do not show at what exact timepoint germ cells become immune to the malignant transformation from these oncogenic events. A temporally controlled, germ cell specific, CreER would allow separation of timing and oncogenic pathways to dissect the exact window of susceptibility. This data would help elucidate what protective mechanisms allow some oncogenic events to be dramatically more efficient at transforming germ cells than others. Currently, we have incorporated the conditional oncogenic alleles of Pten and Kras described above, with the tdTomato reporter, also described above. This additional tracking of early and late malignant cells will allow us to observe the earliest signs of germ cell tumorigenesis in vivo, and also determine DDR and changes in transcriptional markers relative to non-Stra8-cre expressing germ cells at that time. These data will provide additional insight into the timing malignant transformation susceptibility as well as a better understanding of initial malignant changes that occur in these initiating tumors, thus helping better understand their seemingly disparate path of tumorigenesis compared to most somatic cancers.

The next step for these studies is to further elucidate the mechanistic basis of the DDR following cisplatin treatment as well as other genotoxic drugs. P21 expression was shown to be prognostic of chemoresistant EC in humans, likely avoiding the apoptotic response to cisplatin- induced damage through cell cycle arrest, resulting in a refractory response to the treatment123_{. It}

is not yet known if cytosolic P21 levels are abnormally low in chemosensitive cells, but quantifying P21 in our EC lines compared to differentiated TR3 and TR11 cell lines will be informative. It is also not known if these EC cells will exhibit any predicted mechanisms of chemosensitivity such as lacking G1 checkpoint or harboring primed BAX at the golgi, as is reported in mouse embryonic stem cells. Elucidation of these phenotypes within the context of this model requires further

135

experiments to examine cell cycle checkpoint activation, lysate analysis, and response kinetics following exposure to cisplatin discussed further in Chapter 3.

Additionally, it is important to look for response pathways that have yet to be predicted as the underlying cause of chemosensitivity in TGCTs. To do this, genome-wide analysis of mRNA expression is the next immediate step of this study. Previous studies have sought to identify determinants, and the underlying mechanisms, of cisplatin-resistant cancer cells using high- throughput RNA sequencing, but no such studies exist to identify hypersensitivity of EC cells to chemotherapy242,243_{. Beyond the expected pathways of cisplatin sensitivity, this method may reveal}

unexpected pathways that to help unravel the crosstalk between the pluripotent transcriptional network and DDR. This unique approach could lead to a better understanding of these mechanisms and may lead to entirely novel targets for CSCs in somatic cancers.

136 APPENDIX

APPENDIX A. PATIENT DERIVED XENOGRAFTS OF CANINE LYMPHOMA