Summary Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor, AZD1480, suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using shRNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.

SUMMARYMissense mutations in p53 generate aberrant proteins with abrogated tumor suppressor functions that can also acquire oncogenic gain-of-functions (GOF) that promote malignant progression, invasion, metastasis and chemoresistance1–5. Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumors, which is the key requisite for GOF6–8. Although currently 11 million patients worldwide live with tumors expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo.Here we use a novel mutp53 mouse model expressing an inactivatible R248Q hotspot mutation (floxQ) to show that tumors depend on sustained mutp53 expression. Upon Tamoxifen-induced mutp53 ablation, allo-transplanted and autochthonous tumors curb their growth, thus extending animal survival by 37%, and advanced tumors undergo apoptosis and tumor regression or stagnation.The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared to normal tissues, is a major determinant of mutp53 stabilization9–12. We show that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/−2 and H/H (R172H allele3) mice by 59% and 48%, respectively, but not their respective p53−/− littermates. This mutp53-dependent drug effect occurs in H/H mice treated with 17DMAG+SAHA and in H/H and Q/− mice treated with the potent Hsp90 inhibitor ganetespib. Notably, drug activity correlates with induction of mutp53 degradation, tumor apoptosis and prevention of T-lymphomagenesis. These proof-of-principle data identify mutp53 as an actionable cancer-specific drug target.

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Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 mm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first-and second-generation Hsp90 inhibitors. Mol Cancer Ther; 11(2); 475-84. Ó2011 AACR.

EML4-ALK gene rearrangements define a unique subset of non-small cell lung cancer (NSCLC) patients and the clinical success of the ALK inhibitor crizotinib in this population has become a paradigm for molecularly-targeted therapy. Here we show that the Hsp90 inhibitor ganetespib induced loss of EML4-ALK expression and depletion of multiple oncogenic signaling proteins in ALK-driven NSCLC cells, resulting in greater in vitro potency, superior antitumor efficacy and prolonged animal survival compared to crizotinib. In addition, combinatorial benefit was seen when ganetespib was used with other targeted ALK agents both in vitro and in vivo. Importantly, ganetespib overcame multiple forms of crizotinib resistance, including secondary ALK mutations, consistent with activity seen in a NSCLC patient with crizotinib-resistant disease. Cancer cells driven by ALK amplification and oncogenic rearrangements of ROS1 and RET kinases were also sensitive to ganetespib exposure. Taken together, these results highlight the therapeutic potential of ganetespib for ALK-driven NSCLC.

Numerous hormonal factors contribute to the lifetime risk of breast cancer development. These include inherited genetic mutations, age of menarche, age of menopause, and parity. Inexplicably, there is evidence indicating that ovariectomy prevents the formation of both estrogen receptor (ER)-positive and ER-negative breast cancers, suggesting that ERnegative breast cancers are dependent on ovarian hormones for their formation. To examine the mechanism(s) by which this may be occurring, we investigated the hypothesis that steroid hormones promote the outgrowth of ER-negative cancers by influencing host cell types distinct from the mammary epithelial cells. We used a novel xenograft mouse model of parturition-induced breast carcinoma formation, in which the tumors that arise following pregnancy lack the expression of nuclear hormone receptors, thereby recapitulating many clinical cases of this disease. Despite lacking ER expression, the tumors arising following pregnancy in this model require circulating estrogens for their formation. Moreover, increasing the levels of circulating estrogens sufficed to promote the formation and progression of ERnegative cancers, which was accompanied by a systemic increase in host angiogenesis and was attendant with the recruitment of bone marrow-derived stromal cells. Furthermore, bone marrow cells from estrogen-treated mice were sufficient to promote tumor growth. These results reveal a novel mechanism by which estrogens promote the growth of ER-negative cancers. [Cancer Res 2007;67(5):2062-71]

Establishing a model system that more accurately recapitulates both normal and neoplastic breast epithelial development in rodents is central to studying human breast carcinogenesis. However, the inability of human breast epithelial cells to colonize mouse mammary fat pads is problematic. Considering that the human breast is a more fibrous tissue than is the adipose-rich stroma of the murine mammary gland, our group sought to bypass the effects of the rodent microenvironment through incorporation of human stromal fibroblasts. We have been successful in reproducibly recreating functionally normal breast tissues from reduction mammoplasty tissues, in what we term the human-in-mouse (HIM) model. Here we describe our relatively simple and inexpensive techniques for generating this orthotopic xenograft model. Whether the model is to be applied for understanding normal human breast development or tumorigenesis, investigators with minimal animal surgery skills, basic cell culture techniques and access to human breast tissue will be able to generate humanized mouse glands within 3 months. Clearing the mouse of its endogenous epithelium with subsequent stromal humanization takes 1 month. The subsequent implantation of co-mixed human epithelial cells and stromal cells occurs 2 weeks after humanization, so investigators should expect to observe the desired outgrowths 2 months afterward. As a whole, this model system has the potential to improve the understanding of crosstalk between tissue stroma and the epithelium as well as factors involved in breast stem cell biology tumor initiation and progression.

The efficacy of hormonal therapies for advanced estrogen receptorpositive breast cancers is limited by the nearly inevitable development of acquired resistance. Efforts to block the emergence of resistance have met with limited success, largely because the mechanisms underlying it are so varied and complex. Here, we investigate a new strategy aimed at the very processes by which cancers evolve resistance. From yeast to vertebrates, heat shock protein 90 (HSP90) plays a unique role among molecular chaperones by promoting the evolution of heritable new traits. It does so by regulating the folding of a diverse portfolio of metastable client proteins, many of which mediate adaptive responses that allow organisms to adapt and thrive in the face of diverse challenges, including those posed by drugs. Guided by our previous work in pathogenic fungi, in which very modest HSP90 inhibition impairs resistance to mechanistically diverse antifungals, we examined the effect of similarly modest HSP90 inhibition on the emergence of resistance to antiestrogens in breast cancer models. Even though this degree of inhibition fell below the threshold for proteotoxic activation of the heat-shock response and had no overt anticancer activity on its own, it dramatically impaired the emergence of resistance to hormone antagonists both in cell culture and in mice. Our findings strongly support the clinical testing of combined hormone antagonist-low-level HSP90 inhibitor regimens in the treatment of metastatic estrogen receptor-positive breast cancer. At a broader level, they also provide promising proof of principle for a generalizable strategy to combat the pervasive problem of rapidly emerging resistance to molecularly targeted therapeutics.estrogen receptor | antiestrogen | drug resistance | tumor progression | tamoxifen D rastically limiting the efficacy of targeted therapeutics, the emergence of drug resistance in advanced cancers remains nearly inevitable. From yeast to vertebrates, the molecular chaperone heat shock protein 90 (HSP90) allows organisms to adapt and thrive in the face of diverse challenges, including those posed by drugs and environmental stressors (1, 2). It does so by regulating the folding of a highly diverse portfolio of metastable client proteins, many mediating adaptive responses (3, 4).However, this role for HSP90 in adaptation is greatly magnified by its ability to promote the evolution of heritable new traits. To buffer the proteome against unexpected environmental challenges, HSP90 is present in large excess under normal circumstances. This buffering capacity allows it to modulate the manifestation of preexisting and newly acquired genetic variation within heterogeneous populations of cells, and even whole organisms, thereby dramatically expanding the range of phenotypes on which selection can act (1, 2, 5-7). As a dramatic, therapeutically relevant example, we have shown that this HSP90 buffer enables fungal pathogens spanning ∼1 billion years of evolution to evolve and maintain resistance to every major a...