Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in medulloblastoma. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for treating this disease. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed distinct resistance mechanisms. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, or more rarely point mutations in Smo led to reactivated Hh signaling and restored tumor growth. Unexpectedly, analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinositide 3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we demonstrated that the combination of NVP-LDE225 with the PI3K class I inhibitor NVP-BKM120 or the dual PI3K/mTOR inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma.

D-type cyclins (D1, D2 and D3) together with their associated cyclin-dependent kinases CDK4 and CDK6 are components of the core cell cycle machinery that drives cell proliferation1,2. Inhibitors of CDK4 and CDK6 are currently in clinical trials for patients with several cancer types, with promising results2. Here, we show that cyclin D3-CDK6 phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumor cells reduces PPP and serine pathway flows, thereby depleting anti-oxidants NADPH and glutathione. This, in turn elevates the levels of reactive oxygen species and causes tumor cell apoptosis. The pro-survival function of cyclin D-associated kinase operates in tumors expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumor subsets that undergo cell death and tumor regression upon CDK4/6-inhibition. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism represents a particularly powerful oncogene that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.

The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic highthroughput screen was performed, and the lead structure (1) was identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure-activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4 0 -(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.

The link between basal cell carcinoma (BCC) and aberrant activation of the Hedgehog (Hh) signaling pathway has been well established in humans and in mouse models. Here we report the development of assays, including two novel in vitro BCC models, which allowed us to screen for Hh inhibitors and test their validity as potential treatments for BCC. We identified a novel small molecule Hh inhibitor (CUR61414) that can block elevated Hh signaling activity resulting from oncogenic mutations in Patched-1. Moreover, CUR61414 can suppress proliferation and induce apoptosis of basaloid nests in the BCC model systems, whereas having no effect on normal skin cells. These findings directly demonstrate that the use of Hh inhibitors could be a valid therapeutic approach for treating BCC

Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.

Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway.DOI: http://dx.doi.org/10.7554/eLife.18970.001

The ability to create patient derived xenografts (PDXs) has evolved considerably from the breakthrough of the development of immune compromised mice. How researchers in drug discovery have utilized PDX of certain cancer types has also changed from traditionally selecting a few models to profile a drug, to opting to assess inter-tumor response heterogeneity by screening across a broad range of tumor models, and subsequently to enable clinical stratification strategies. As with all models and methodologies, imperfections with this approach are apparent, and our understanding of the fidelity of these models continues to expand. To date though, they are still viewed as one of the most faithful modeling systems in oncology. Currently, there are many efforts ongoing to increase the utility and translatability of PDXs, including introducing a human immune component to enable immunotherapy studies.