Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from, a crizotinib treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076), and from studying a resistant version of the ALK TKI (TAE684) sensitive H3122 cell line. The crizotinib resistant DFCI076 cell line, harboured a unique L1152R ALK secondary mutation, and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent co-activation of epidermal growth factor receptor (EGFR) signalling. In contrast, the TAE684 resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harboured co-activation of EGFR signalling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naïve NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signalling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.

CD200 (OX-2) is a cell surface glycoprotein that imparts immune privileges by suppressing alloimmune and autoimmune responses through its receptor, CD200R, expressed primarily on myeloid cells. The ability of CD200 to suppress myeloid cell activation is critical for maintaining normal tissue homeostasis but may also enhance the survival of migratory neoplastic cells. We show that CD200 expression is largely absent in welldifferentiated primary squamous cell carcinoma (SCC) of the skin, but is highly induced in SCC metastases to the lymph node and other solid tissues. CD200 does not influence the proliferative or invasive capacity of SCC cells or their ability to reconstitute primary skin tumors. However, loss of CD200 impairs the ability of SCC cells to metastasize and seed secondary tumors, indicating that the survival of CD200 + SCC cells may depend on their ability to interact with CD200R + immune cells. The predominant population of CD200R + stromal cells was CD11b + Gr-1 + myeloid-derived suppressor cells, which release elevated levels of granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor when in the presence of SCC cells in a CD200-dependent manner. Collectively, our findings implicate CD200 as a hallmark of SCC metastasis and suggest that the ability of CD200 + SCC keratinocytes to directly engage and modulate CD200R + myeloid-derived suppressor cells is essential to metastatic survival. Cancer Res; 70(7); 2962-72. ©2010 AACR.

Epithelial stem cells in adult mammalian skin are known to maintain epidermal, follicular and sebaceous lineages during homeostasis. Recently, Merkel cell mechanoreceptors were identified as a fourth lineage derived from the proliferative layer of murine skin epithelium; however, the location of the stem or progenitor population for Merkel cells remains unknown. Here, we have identified a previously undescribed population of epidermal progenitors that reside in the touch domes of hairy skin, termed touch dome progenitor cells (TDPCs). TDPCs are epithelial keratinocytes and are distinguished by their unique co-expression of α6 integrin, Sca1 and CD200 surface proteins. TDPCs exhibit bipotent progenitor behavior as they give rise to both squamous and neuroendocrine epidermal lineages, whereas the remainder of the α6+ Sca1+ CD200– epidermis does not give rise to Merkel cells. Finally, TDPCs possess a unique transcript profile that appears to be enforced by the juxtaposition of TDPCs with Merkel cells within the touch dome niche.

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Rimkunas and colleagues report on the development and validation of an immunohistochemical assay evaluating non-small cell lung cancers (NSCLC) for the presence of ROS1 fusions. The diagnostic was validated in a screen of 556 NSCLCs, identifying 9 (1.6%) tumors with oncogenic ROS1 rearrangements. These patients are candidates for ROS1-targeted therapies. Clin Cancer Res; 18(16); 4222-4. Ó2012 AACR.In this issue of Clinical Cancer Research, Rimkunas and colleagues report on a novel immunohistochemical assay designed to identify the expression of ROS1. Using this assay, the authors identified a small but therapeutically significant percentage of non-small cell lung cancers (NSCLC) harboring genomic ROS1 rearrangements (1).ROS1, a gene coding for a receptor tyrosine kinase of the insulin receptor family, is involved in chromosomal translocations in a number of cancers (2-4) and has recently been shown to undergo a rearrangement leading to the formation of a fusion oncogene in NSCLC (refs. 5, 6; Fig. 1). ROS1 chromosomal rearrangements were first described in glioblastomas, where ROS1 is fused with FIG as a result of an interstitial chromosomal deletion (3), and the chimeric protein was confirmed to be oncogenic in a transgenic mouse model (7). Very recently, a number of novel ROS1 fusion and translocation products have been identified as potential oncogenic drivers in primary NSCLC tumors ( Fig. 1) and in the HCC78 cell line harboring the SLC34A2-ROS1 fusion (1,5,6). Within these chimeric proteins, ROS1 is fused with its partners at exons 32, 34, 35, or 36 of ROS1. These breakpoints allow for the retention of the ROS1 kinase domain, constitutive kinase activity, and inferred transforming potential. Activation of the ROS1 kinase is brought about by the dimerization of the ROS1 fusion mediated by the N-terminal fusion partner. Subsequently, the activated ROS1 kinase stimulates downstream signaling, resulting in enhanced cell growth, proliferation, and decreased apoptosis.To date, ROS1 rearrangements are not found to overlap with other oncogenic mutations commonly detected in NSCLC, including EGF receptor (EGFR) mutations, KRAS mutations, or ALK rearrangements (5, 8). Preclinical studies indicate that ROS1 fusion harboring tumors and cell lines are sensitive to the dual ALK/MET inhibitor crizotinib, an observation also confirmed by a partial response of a patient with treated CD74-ROS1-positive NSCLC to crizotinib therapy and more recently in a larger cohort of patients with NSCLC harboring ROS1 rearrangements (8, 9). Furthermore, preclinical studies show that cells harboring ROS1 fusions do not respond to treatment with EGFRtargeted kinase inhibitors. These preliminary results define ROS1-rearranged NSCLC as a discrete druggable entity and underscore the importance of conclusive identification of ROS1-rearranged patients with lung cancer who may benefit from ROS1-targeted therapy.In this issue, Rimkunas and colleagues describe a highly sensitive and specific ROS1 antibody developed to analyze ROS1 fusion expression ...

Supplementary Figure 3 from The Immunosuppressive Surface Ligand CD200 Augments the Metastatic Capacity of Squamous Cell Carcinoma

Supplementary Table 1 from The Immunosuppressive Surface Ligand CD200 Augments the Metastatic Capacity of Squamous Cell Carcinoma

Supplementary Materials and Methods from The Immunosuppressive Surface Ligand CD200 Augments the Metastatic Capacity of Squamous Cell Carcinoma