Stanley Lipkowitz

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

The Death Domain Kinase RIP Is Essential for TRAIL (Apo2L)-Induced Activation of IκB Kinase and c-Jun N-Terminal Kinase

ABSTRACT Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-κB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IκB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559–671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.

Triple negative breast cancer cell lines: One tool in the search for better treatment of triple negative breast cancer

Breast cancer is the most frequently diagnosed cancer in women and one of the leading causes of cancer death for women. Worldwide, over 1.3 million cases of invasive breast cancer are diagnosed, and more than 450,000 women die from breast cancer annually [1]. In the US, approximately 200,000 cases of invasive breast cancer and 50,000 cases of in situ breast cancer will be diagnosed annually, and more than 40,000 women die from breast cancer each year – second only to lung cancer [2]. The mortality due to breast cancer has been declining in the US since 1990 [2]. The continuing decrease in mortality from breast cancer has been attributed to early detection due to screening, improved adjuvant therapy, and more recently to decreases in the incidence due to lowered rates of usage of hormone replacement therapy [3, 4]. Despite the decreased incidence and mortality, breast cancer remains a major cause of cancer mortality for women and accounts for 15% of all cancer deaths in women in the US [2]. Clinically, breast cancer can be divided into distinct subtypes that have prognostic and therapeutic implications. Breast cancer patients routinely have the expression of estrogen receptor (ER), progesterone receptor (PR), and amplification of HER-2/Neu evaluated [5]. These markers allow classification of breast cancer tumors as hormone receptor positive tumors, HER-2/Neu amplified tumors, and those tumors which do not express ER, PR, and do not have HER-2/Neu amplification. The latter group is referred to as triple-negative breast cancer (TNBC) based on the lack of these three molecular markers. Generally, hormone receptor expressing breast cancers have a more favorable prognosis than either those with HER-2/Neu amplification or those that are triple-negative [5]. While all breast tumor types may be treated with chemotherapy, therapeutic options in both early and late stage breast cancer are affected significantly by the expression of these three markers. Tumors that express ER and PR are treated with agents that interfere with hormone production or action [5]. Tumors that have amplified HER-2/Neu are treated with agents that inhibit HER-2/Neu [5]. These targeted therapies are the mainstay of the successful outcomes seen in hormone receptor positive and HER-2/Neu amplified tumors. Both early stage and advanced TNBC tumors are treated with predominantly chemotherapy [5]. TNBC represents approximately 10–15% of all breast cancers and patients with TNBC have a poor outcome compared to the other subtypes of breast cancer [6]. Interestingly, the incidence of TNBC in African American women is two to three times higher than other ethnic groups, although the reason for this has not been elucidated [6, 7]. Given the lack of validated molecular targets and the poor outcome in patients with TNBC, there is a clear need for a greater understanding of TNBC at all levels and for the development of better therapies. Cancer cell lines have proved useful in laboratory and preclinical investigations since the first cell line was established more than 50 years ago [8]. For example, the anti-HER-2/Neu 4D5 mouse monoclonal antibody (which was humanized to create trastuzumab) had anti-tumor effects as a single agent and acted synergistically with chemotherapeutic agents in breast cancer cell lines that had amplified HER-2/Neu but showed no effect in cell lines lacking amplified HER-2/Neu [9–12]. These results formed the basis for clinical trials of trastuzmab and predicted the outcomes of these clinical trials. Similarly, preclinical studies using xenografts of a hormone receptor expressing breast cancer cell line have accurately predicted the outcomes of clinical trials comparing aromatase inhibitors to tamoxifen and to the combination of tamoxifen and aromatase inhibitors [13]. In this review, we will describe triple negative breast cancer cell lines and discuss their utility and the limitations of these cell lines in the investigation of TNBC (mindful of Harry Callahan’s admonition cited above).

Cbl and human myeloid neoplasms: the Cbl oncogene comes of age

Cbl was originally discovered in 1989 as the cellular homolog of the v-Cbl oncogene, the transforming gene of the Cas NS-1 murine retrovirus that causes myeloid leukemia and lymphomas in mice. Cbl is a member of a family of RING finger ubiquitin ligases that negatively regulate signaling by tyrosine kinases and that function as adaptor proteins to regulate signaling positively. Until the past 2 years, there was little evidence that Cbl proteins were involved in human malignancies. Recent publications have shown homozygous mutations in Cbl in human myeloid neoplasms. Although in vitro and animal transformation models suggested that mutant forms of Cbl acted as an oncogene, the cellular role suggested that the protein could serve as a tumor suppressor gene. The recent data begin to reconcile this paradox as the loss of ubiquitin ligase function (the tumor suppressor function) is coupled to the maintenance of the positive signaling function (the oncogene function). These data also provide insight into potential therapeutic approaches to myeloid disorders harboring Cbl mutations.

The N Terminus of Cbl-c Regulates Ubiquitin Ligase Activity by Modulating Affinity for the Ubiquitin-conjugating Enzyme

Cbl proteins are ubiquitin ligases (E3s) that play a significant role in regulating tyrosine kinase signaling. There are three mammalian family members: Cbl, Cbl-b, and Cbl-c. All have a highly conserved N-terminal tyrosine kinase binding domain, a catalytic RING finger domain, and a C-terminal proline-rich domain that mediates interactions with Src homology 3 (SH3) containing proteins. Although both Cbl and Cbl-b have been studied widely, little is known about Cbl-c. Published reports have demonstrated that the N terminus of Cbl and Cbl-b have an inhibitory effect on their respective E3 activity. However, the mechanism for this inhibition is still unknown. In this study we demonstrate that the N terminus of Cbl-c, like that of Cbl and Cbl-b, inhibits the E3 activity of Cbl-c. Furthermore, we map the region responsible for the inhibition to the EF-hand and SH2 domains. Phosphorylation of a critical tyrosine (Tyr-341) in the linker region of Cbl-c by Src or a phosphomimetic mutation of this tyrosine (Y341E) is sufficient to increase the E3 activity of Cbl-c. We also demonstrate for the first time that phosphorylation of Tyr-341 or the Y341E mutation leads to a decrease in affinity for the ubiquitin-conjugating enzyme (E2), UbcH5b. The decreased affinity of the Y341E mutant Cbl-c for UbcH5b results in a more rapid turnover of bound UbcH5b coincident with the increased E3 activity. These data suggest that the N terminus of Cbl-c contributes to the binding to the E2 and that phosphorylation of Tyr-341 leads to a decrease in affinity and an increase in the E3 activity of Cbl-c.

ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking.

Background information. ARAP1 is an Arf (ADP‐ribosylation factor)‐directed GAP (GTPase‐activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1.

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.

Prexasertib, a cell cycle checkpoint kinase 1 and 2 inhibitor, in BRCA wild-type recurrent high-grade serous ovarian cancer: a first-in-class proof-of-concept phase 2 study

Background High-grade serous ovarian carcinoma (HGSOC) is characterized by TP53 mutations, DNA repair defects, and genomic instability. We hypothesized that prexasertib, a cell cycle checkpoint kinase 1 and 2 inhibitor, would be active in BRCA wild-type HGSOC. Methods In this open label, single centre, two-stage proof-of-concept phase 2 study, women aged 18 years or older with measurable, recurrent high-grade serous or high-grade endometrioid ovarian carcinoma were enrolled. All patients must have had either a negative family history of hereditary breast and ovarian cancer or known BRCA wild-type for BRCA wild-type cohort. Other key eligibility criteria were an Eastern Cooperative Oncology Group performance status of 0 or 1 or 2, and adequate haematological, renal, and hepatic function. Patients received intravenous prexasertib 105mg/m2 once every 2 weeks until disease progression, unacceptable toxicity or patient withdrawal of consent. The primary endpoint was investigator-assessed tumour response per protocol based on Response Evaluation Criteria in Solid Tumors, version 1·1 in evaluable patients. The final analysis of this cohort is reported here. This ongoing trial is registered with ClinicalTrials.gov (NCT02203513) and enrolling the patients of BRCA mutation cohort. Findings Between January 2015 and November 2016, 28 women (median age 64-year-old [IQR 58–69·5], with median 5 prior systemic therapies [IQR 2·5–5]) were enrolled and received at least one dose of prexasertib. Eight of 24 evaluable patients had a partial response (PR; 33%, 95% CI: 16–55) and 50% had a GCIG CA125 response. The RR in the intention-to-treat population was 29% (8/28, 95% CI: 13–49). The common (>10%) grade 3 or 4 treatment-emergent adverse events were neutropenia (26 [93%] patients), thrombocytopenia (seven [25%] patients), and anaemia (three [11%] patients). Grade 4 neutropenia occurred in 22 (79%) patients after the first dose and was transient ≤ 7 days (median 6 days [IQR 4–8]) without growth factor support; the incidence of febrile neutropenia was 7% (2/28). Interpretation We demonstrate clinical activity of prexasertib in BRCA wild-type HGSOC, especially patients with platinum-resistant or refractory ovarian cancer. These results warrant further development for this unmet patient need. Funding Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research, USA.

ONC201 kills breast cancer cells in vitro by targeting mitochondria

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Abstract 15: gp78 is a negative regulator of TRAIL-induced apoptosis in breast cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in transformed cells by activating the extrinsic apoptotic pathway via its cognate receptors on the cell surface, TRAIL receptor 1 and TRAIL receptor 2. Triple negative breast cancer (TNBC) cells (so-called because TNBC lacks estrogen and progesterone receptor expression and Her-2 amplification) have been found to be sensitive to TRAIL while breast cancer cells of other subtypes of disease remain relatively resistant. Unfortunately, the mechanisms that govern sensitivity to TRAIL are not yet understood. The identification and characterization of novel regulators of the TRAIL pathway would provide new insights into the mechanisms that regulate TRAIL and potentially provide therapeutically exploitable molecular targets for the enhancement of TRAIL-based cancer treatments. In order to identify candidate regulators of the TRAIL pathway, our lab carried out a high-throughput RNAi-mediated screen of ∼1300 genes using the TNBC cell line MB231. One hundred fifty candidate regulators were identified. The RING finger ubiquitin ligase gp78 (also known as AMFR) was identified as a candidate negative regulator of TRAIL sensitivity. gp78 is an endoplasmic reticulum (ER)-residing protein that helps facilitate the retrotranslocation of substrates across the ER membrane into the cytosol during ER-associated protein degradation. This process is critical to maintaining cellular homeostasis by promoting the proteasomal elimination of misfolded proteins and is integral to cell survival. Interestingly, gp78 has previously been found to promote metastasis in a mouse sarcoma model, and in this study, we have further characterized gp78 as an inhibitor of apoptosis. The initial findings from the RNAi screen were confirmed by carrying out siRNA-mediated knockdown of gp78 in MB231 cells. The cells with inhibited gp78 expression were found to be significantly sensitive to TRAIL-induced caspase-3/7 activity and loss in viability. Knockdown of gp78 using in total 11 independent siRNAs demonstrated a reduction in gp78 expression is associated with TRAIL sensitization. Furthermore, pan-caspase inhibition with the pharmacologic inhibitor ZVAD-FMK completely abrogated sensitization to TRAIL with gp78 knockdown, demonstrating that loss in viability is caspase dependent. These results were further characterized by knocking down gp78 alone or along with one of the initiator caspases, 2, 8, 9, or 10. Loss of the initiator caspases 2 and 8 reduced sensitivity to TRAIL with gp78 knockdown, suggesting that pathways involved in the activation of these caspases in particular may be conferring sensitivity to TRAIL. In summary, our lab has identified and characterized gp78 as a regulator of TRAIL sensitivity through a caspase-mediated mechanism in breast cancer cells. Continued study is warranted to fully elucidate the molecular mechanisms by which gp78 inhibits TRAIL-induced apoptosis. Citation Format: Jennifer L. Dine, Sireesha V. Garimella, Kristie Gehlhaus, Magda Grandin, Daniel Letwin, Natasha Caplen, Stanley Lipkowitz. gp78 is a negative regulator of TRAIL-induced apoptosis in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 15. doi:10.1158/1538-7445.AM2015-15