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Summary NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.

// Kumar Saurabh 1,* , Michael T. Scherzer 1,4,* , Parag P. Shah 1 , Alice S. Mims 5 , William W. Lockwood 6 , Andrew S. Kraft 5 and Levi J. Beverly 1,2,3,4 1 James Graham Brown Cancer Center, University of Louisville, Louisville, KY 2 Department of Medicine, Division of Hematology and Oncology, University of Louisville School of Medicine, Louisville, KY 3 Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 4 Department of Bioengineering, J.B Speed School of Engineering, University of Louisville, Louisville, KY 5 Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 6 Integrative Oncology, British Columbia Cancer Agency, Vancouver, British Columbia, Canada * These authors contributed equally to this work Correspondence: Levi J. Beverly, email: // Keywords : PIM-1, PIM-2, PIM-3, MYC, leukemia Received : July 08, 2014 Accepted : August 07, 2014 Published : August 08, 2014 Abstract PIM kinases are a family of serine/threonine kinases involved in cell survival and proliferation. There is significant structural similarity between the three PIM kinases (PIM1, PIM2 and PIM3) and only few amino acid differences. Although, several studies have specifically monitored the role of PIM1 in tumorigenesis, much less is known about PIM2 and PIM3. Therefore, in this study we have used in vitro cell culture models and in vivo bone marrow infection/transplantation to assess the comparative signaling and oncogenic potential of each of the three PIM kinases. All three PIM kinases were able to protect FL5.12 cells from IL3 withdrawal induced death. Interestingly, the downstream signaling cascades were indistinguishable between the three kinases. Transplantation of murine bone marrow co-expressing MYC and PIM1, PIM2 or PIM3 caused rapid and uniformly lethal myeloid leukemia. De-induction of MYC 18 days following transplantation significantly increased the survival of mice, even with continual expression of PIM kinases. Alternatively, mice treated at the pre-leukemic stage with a PIM kinase inhibitor increased the lifespan of the mice, even with continual expression of the MYC transgene. These data demonstrate the role of PIM kinases in driving myeloid leukemia, and as candidate molecules for therapy against human malignancies.

Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.

Overexpression of anti-apoptotic members of the BCL2 family has been found in all types of cancer. A member of the family, BCLxl (B-cell lymphoma extra-large), is known to be associated with the progression of leukemogenesis. In the present study, we focused on understanding the domains of BCLxl responsible for in vivo oncogenic potency. To this end, we utilized engineered BCLxl proteins with alternative transmembrane domains (TM) or chimeric BCLxl proteins containing domains from a less potent BCL2-like protein, BCLb. As expected, mice receiving MYC-only expressing bone marrow develop leukemia by 100 days, whereas co-expression of MYC with wild-type BCLxl led to aggressive myeloid leukemia with an average latency of ~25 days. Interestingly, mice injected with bone marrow co-expressing MYC and BCLxl targeted specifically to either mitochondria or ER also succumbed to leukemia with an average latency of ~25 days. Further, our study was extended to examine the role of the BH4 domain in driving potent leukemogenesis. Mice injected with bone marrow co-expressing MYC and BCLb succumb to leukemia in an average of ~55 days, but interestingly a BCLxl protein containing only the loop region of BCLb drove MYC-induced leukemogenesis with the same latency as wild-type BCLxl. These data suggest that the localization of exogenous BCLxl to either mitochondria or ER is not a steadfast dictator of in vivo oncogenic potency. Further, our findings suggest that the loop domain of BCLb and BCLxl is not responsible for dictating the in vivo leukemogeneic potency. This study provides further mechanistic details into the biochemical functions of BCLxl.