Edward Rosfjord

Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase

HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25–30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Trastuzumab (Herceptin), a monoclonal antibody to HER-2, is specifically approved for HER-2-positive breast cancer but is active only in a subset of these tumors. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors. HKI-272 is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. HKI-272 reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G1-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.

Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression

SummaryWe have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1–S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.

Advances in patient-derived tumor xenografts: From target identification to predicting clinical response rates in oncology

Most oncology compounds entering clinical development have passed stringent preclinical pharmacology evaluation criteria. However, only a small fraction of experimental agents induce meaningful antitumor activities in the clinic. Low predictability of conventional preclinical pharmacology models is frequently cited as a main reason for the unusually high clinical attrition rates of therapeutic compounds in oncology. Therefore, improvement in the predictive values of preclinical efficacy models for clinical outcome holds great promise to reduce the clinical attrition rates of experimental compounds. Recent reports suggest that pharmacology studies conducted with patient derived xenograft (PDX) tumors are more predictive for clinical outcome compared to conventional, cell line derived xenograft (CDX) models, in particular when therapeutic compounds were tested at clinically relevant doses (CRDs). Moreover, the study of the most malignant cell types within tumors, the tumor initiating cells (TICs), relies on the availability of preclinical models that mimic the lineage hierarchy of cells within tumors. PDX models were shown to more closely recapitulate the heterogeneity of patient tumors and maintain the molecular, genetic, and histological complexity of human tumors during early stages of sequential passaging in mice, rendering them ideal tools to study the responses of TICs, tumor- and stromal cells to therapeutic intervention. In this commentary, we review the progress made in the development of PDX models in key areas of oncology research, including target identification and validation, tumor indication search and the development of a biomarker hypothesis that can be tested in the clinic to identify patients that will benefit most from therapeutic intervention.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions

PTK7 is a tumor-initiating cell antigen, which can be targeted with an antibody-drug conjugate to confer sustained tumor regressions. Initiating an antitumor attack Cancer is notorious for relapsing after treatment, making it difficult to eradicate from a patient’s body. Such relapses are driven by tumor-initiating cells, a type of stem cells that give rise to tumors. Damelin et al. determined that a protein called PTK7 is frequently present on tumor-initiating cells and developed an antibody-drug conjugate for targeting it. The authors demonstrated the effectiveness of this therapy in mouse models of several tumor types and confirmed that it reduces tumor-initiating cells and outperforms standard chemotherapy. The antibody-drug conjugate also had some unexpected benefits, reducing tumor angiogenesis and promoting antitumor immunity, all of which may contribute to its effectiveness. Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non–small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient–derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline–based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Control of mammary tumor differentiation by SKI-606 (bosutinib)

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

The interaction of PKN3 with RhoC promotes malignant growth

PKN3 is an AGC‐family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3‐kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in‐vitro. Mechanistically, we demonstrate that PKN3 physically interacts with Rho‐family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late‐stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co‐expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3–RhoC complex represents an attractive therapeutic target in late‐stage malignancies.

p120 catenin is a key effector of a Ras-PKCɛ oncogenic signaling axis

Within the family of protein kinase C (PKC) molecules, the novel isoform PRKCE (PKCɛ) acts as a bona fide oncogene in in vitro and in vivo models of tumorigenesis. Previous studies have reported expression of PKCɛ in breast, prostate and lung tumors above that of normal adjacent tissue. Data from the cancer genome atlas suggest increased copy number of PRKCE in triple negative breast cancer (TNBC). We find that overexpression of PKCɛ in a non-tumorigenic breast epithelial cell line is sufficient to overcome contact inhibition and results in the formation of cellular foci. Correspondingly, inhibition of PKCɛ in a TNBC cell model results in growth defects in two-dimensional (2D) and three-dimensional (3D) culture conditions and orthotopic xenografts. Using stable isotope labeling of amino acids in a cell culture phosphoproteomic approach, we find that CTNND1/p120ctn phosphorylation at serine 268 (P-S268) occurs in a strictly PKCɛ-dependent manner, and that loss of PKCɛ signaling in TNBC cells leads to reversal of mesenchymal morphology and signaling. In a model of Ras activation, inhibition of PKCɛ is sufficient to block mesenchymal cell morphology. Finally, treatment with a PKCɛ ATP mimetic inhibitor, PF-5263555, recapitulates genetic loss of function experiments impairing p120ctn phosphorylation as well as compromising TNBC cell growth in vitro and in vivo. We demonstrate PKCɛ as a tractable therapeutic target for TNBC, where p120ctn phosphorylation may serve as a readout for monitoring patient response.

Enhanced Antitumor Activity of an Anti-5T4 Antibody–Drug Conjugate in Combination with PI3K/mTOR inhibitors or Taxanes

Purpose: Targeted treatment of solid or liquid tumors with antibody–drug conjugates (ADCs) can lead to promising clinical benefit. The aim of the study is to investigate combination regimens of auristatin-based ADCs in preclinical models of cancer. Experimental Design: An auristatin-based anti-5T4 antibody conjugate (5T4-ADC) and auristatin payloads were combined with the dual PI3K/mTOR catalytic site inhibitor PF-05212384 (PF-384) or taxanes in a panel of tumor cell lines. Drug interactions in vitro were evaluated using cell viability assays, apoptosis induction, immunofluorescence, mitotic index, and immunoblotting. Breast cancer cells treated with auristatin analogue or 5T4-ADC were profiled by total- and phospho-proteomics. Antitumor efficacy of selected combinations was evaluated in 5T4-positive human breast or lung tumor xenografts in vivo. Results: In vitro, auristatin-based agents displayed strong synergistic or additive activity when combined with PF-384 or taxanes, respectively. Further, treatment of 5T4-ADC plus PF-384 resulted in stronger induction of apoptosis and cell line–specific attenuation of pAKT and pGSK. Interestingly, proteomic analysis revealed unique effects of auristatins on multiple components of mRNA translation. Addition of PF-384 further amplified effects of 5T4-ADC on translational components, providing a potential mechanism of synergy between these drugs. In human tumor xenografts, dual targeting with 5T4-ADC/PF-384 or 5T4-ADC/paclitaxel produced substantially greater antitumor effects with longer average survival as compared with monotherapy treatments. Conclusions: Our results provide a biologic rationale for combining 5T4-ADC with either PI3K/mTOR pathway inhibitors or taxanes and suggest that mechanisms underlying the synergy may be attributed to cellular effects of the auristatin payload. Clin Cancer Res; 22(2); 383–94. ©2015 AACR.

Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1)

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1–resistant population was generated. T-DM1–resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non–cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)–positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243–53. ©2017 AACR.

Abstract 2113: Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprised of the HER2-targeting antibody trastuzumab and DM1, a microtubule depolymerizing agent covalently attached via a non-cleavable thioether linker. T-DM1 has demonstrated clinical benefit for patients with metastatic breast cancer, however activity may be limited by inherent or acquired resistance during prolonged treatment periods. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 positive tumors, are not well understood. We used the HER2+ cell line N87 to develop a model of T-DM1 resistance utilizing a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1 resistant population of N87 cells (N87-TM) was generated. N87-TM cells displayed 103-fold resistance toward T-DM1 treatment compared to the parental N87 cells. The N87-TM cells were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug efflux pump (e.g. MDR1) protein expression compared to parental N87 cells. Comparative proteomic profiling suggested an enrichment in proteins (e.g. caveolin-1, CAV1) that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize ADCs into intracellular CAV1+ puncta and alter their trafficking to the lysosome compared to N87 cells. Intriguingly, T-ADCs utilizing auristatin payloads attached via an enzymatically cleavable linker (i.e. ValCit linker) overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice in vivo formed T-DM1 refractory tumors which remain sensitive to T-ADCs with cleavable-linked auristatin payloads. When comparing antigen positive patient-derived xenograft models that were refractory to T-DM1 yet responded to T-ADCs with ValCit linker-payloads, CAV1 was found to be a predictive protein biomarker identifying T-DM1 refractory tumors. These data implicate caveolae-mediated endocytosis in ADC biology and suggest that alterations in this pathway may impact a tumor9s response profile to ADCs with non-cleavable linkers. We also propose CAV1 as a novel protein biomarker whose high tumoral expression predicts a refractory response to the T-DM1 ADC. Citation Format: Matthew S. Sung, Xingzhi Tan, Christine Hosselet, Michael Cinque, Erik Upeslacis, Jonathon Golas, Fang Wang, Bingwen Lu, Laurie Tylaska, Lindsay King, Jeremy Myers, Edward Rosfjord, Judy Lucas, Hans-Peter Gerber, Frank Loganzo. Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2113.