Claire M. Faltermeier

Metastatic castration-resistant prostate cancer reveals intrapatient similarity and interpatient heterogeneity of therapeutic kinase targets

Significance Metastatic castration-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. The need to identify new actionable targets in CRPC is crucial as we begin to examine the resistance mechanisms related to androgen withdrawal. Here, we report an unbiased quantitative phosphoproteomic approach to identify druggable kinases in metastatic CRPC. These kinase activation patterns revealed intrapatient similarity and interpatient heterogeneity across a large panel of targets. Interestingly, these kinase activities are not a result of mutation but rather pathway activation within the tumors themselves. The observation that similar kinase activities are present in most if not all anatomically disparate metastatic lesions from the same patient suggests that CRPC patients may benefit from individualized, targeted combination therapies. In prostate cancer, multiple metastases from the same patient share similar copy number, mutational status, erythroblast transformation specific (ETS) rearrangements, and methylation patterns supporting their clonal origins. Whether actionable targets such as tyrosine kinases are also similarly expressed and activated in anatomically distinct metastatic lesions of the same patient is not known. We evaluated active kinases using phosphotyrosine peptide enrichment and quantitative mass spectrometry to identify druggable targets in metastatic castration-resistant prostate cancer obtained at rapid autopsy. We identified distinct phosphopeptide patterns in metastatic tissues compared with treatment-naive primary prostate tissue and prostate cancer cell line-derived xenografts. Evaluation of metastatic castration-resistant prostate cancer samples for tyrosine phosphorylation and upstream kinase targets revealed SRC, epidermal growth factor receptor (EGFR), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), and MAPK1/3 and other activities while exhibiting intrapatient similarity and interpatient heterogeneity. Phosphoproteomic analyses and identification of kinase activation states in metastatic castration-resistant prostate cancer patients have allowed for the prioritization of kinases for further clinical evaluation.

Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer

We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients.

Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer

Significance A high nuclear Notch homolog 1, translocation-associated (Notch1) intracellular domain level distinguishes high-risk prostate cancer and castration-resistant prostate cancer from benign and low/intermediate-risk prostate cancer. Chronic activation of Notch1 cooperates with multiple oncogenic pathways altered in early prostate cancer, including AKT, Myc, and Ras/Raf/MAPK, to promote progression to androgen ablation-resistant prostate adenocarcinoma. Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

Functional screen identifies kinases driving prostate cancer visceral and bone metastasis

Significance Therapies are urgently needed to treat metastatic prostate cancer. Mutationally activated and wild-type kinases such as BCR-ABL and BTK are effective therapeutic targets in multiple cancers. Genetically altered kinases are rare in prostate cancer. Wild-type kinases may be implicated in prostate cancer progression, but their therapeutic potential in metastatic prostate cancer remains unknown. Using phosphoproteomics and gene expression datasets, we selected 125 wild-type kinases implicated in human prostate cancer metastasis to screen for metastatic ability in vivo. The RAF family, MERTK, and NTRK2 drove prostate cancer bone and visceral metastasis and were highly expressed in human metastatic prostate cancer tissues. These studies reveal that wild-type kinases can drive metastasis and that the RAF family, MERTK, and NTRK2 may represent important therapeutic targets. Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention.

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

Background Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia. Methodology/Principal Findings The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int. Conclusions/Significance Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.

Surgical Technical Evidence Review for Elective Total Joint Replacement Conducted for the AHRQ Safety Program for Improving Surgical Care and Recovery

Background: Use of enhanced recovery pathways (ERPs) can improve patient outcomes, yet national implementation of these pathways remains low. The Agency for Healthcare Research and Quality (AHRQ; funder), the American College of Surgeons, and the Johns Hopkins Medicine Armstrong Institute for Patent Safety and Quality have developed the Safety Program for Improving Surgical Care and Recovery—a national effort to catalyze implementation of practices to improve perioperative care and enhance recovery of surgical patients. This review synthesizes evidence that can be used to develop a protocol for elective total knee arthroplasty (TKA) and total hip arthroplasty (THA). Study Design: This review focuses on potential components of the protocol relevant to surgeons; anesthesia components are reported separately. Components were identified through review of existing pathways and from consultation with technical experts. For each, a structured review of MEDLINE identified systematic reviews, randomized trials, and observational studies that reported on these components in patients undergoing elective TKA/THA. This primary evidence review was combined with existing clinical guidelines in a narrative format. Results: Sixteen components were reviewed. Of the 10 preoperative components, most were focused on risk factor assessment including anemia, diabetes mellitus, tobacco use, obesity, nutrition, immune-modulating therapy, and opiates. Preoperative education, venous thromboembolism (VTE) prophylaxis, and bathing/Staphylococcus aureus decolonization were also included. The routine use of drains was the only intraoperative component evaluated. The 5 postoperative components included early mobilization, continuous passive motion, extended duration VTE prophylaxis, early oral alimentation, and discharge planning. Conclusion: This review synthesizes the evidence supporting potential surgical components of an ERP for elective TKA/THA. The AHRQ Safety Program for Improving Surgical Care and Recovery aims to guide hospitals and surgeons in identifying the best practices to implement in the surgical care of TKA and THA patients.

Surgical Technical Evidence Review of Hip Fracture Surgery Conducted for the AHRQ Safety Program for Improving Surgical Care and Recovery

Background: Enhanced recovery pathways (ERPs) have been shown to improve patient outcomes in a variety of contexts. This review summarizes the evidence and defines a protocol for perioperative care of patients with hip fracture and was conducted for the Agency for Healthcare Research and Quality safety program for improving surgical care and recovery. Study Design: Perioperative care was divided into components or “bins.” For each bin, a semisystematic review of the literature was conducted using MEDLINE with priority given to systematic reviews, meta-analyses, and randomized controlled trials. Observational studies were included when higher levels of evidence were not available. Existing guidelines for perioperative care were also incorporated. For convenience, the components of care that are under the auspices of anesthesia providers will be reported separately. Recommendations for an evidence-based protocol were synthesized based on review of this evidence. Results: Eleven bins were identified. Preoperative risk factor bins included nutrition, diabetes mellitus, tobacco use, and anemia. Perioperative management bins included thromboprophylaxis, timing of surgery, fluid management, drain placement, early mobilization, early alimentation, and discharge criteria/planning. Conclusions: This review provides the evidence basis for an ERP for perioperative care of patients with hip fracture.

Abstract 3882: Patient-specific druggable networks in lethal prostate cancer through proteome-guided multi-omic integration

Metastatic castration resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. The need to identify new actionable targets in this disease is crucial as we begin to examine the resistance mechanisms related to androgen withdrawal. Pathway activation of signaling proteins, such as kinases, are hypothesized to drive the progression of lethal CRPC. We set out to define the global picture of signaling pathways in lethal prostate cancer through dataset integration. We used clinical tissue from lethal metastatic CRPC patients obtained at rapid autopsy to evaluate and integrate previously published genomic and transcriptomic datasets combined with a new complete and extensive dataset of the phosphoproteome in metastatic CRPC for pathway analysis. This included extending our analysis of the phosphoproteome to phosphoserine and phosphothreonine peptides with our published phosphotyrosine peptide data. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues relative to treatment naive prostate cancer to synthesize a robust signaling network consisting of druggable kinase pathways for therapy. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Further, we were able to gather mRNA and phosphoproteome data from six individual patients where the integration of tissue samples from a single autopsy program allowed us to make inferences on the connections between the mRNA and phosphoproteome datasets. Individual patient profiles developed using these hallmarks revealed clinically relevant pathway information suitable for patient stratification and targeted therapies in lethal prostate cancer. Here we describe these pathways: personalized cancer hallmarks using an integrative phospho-signature (pCHIPs) that sheds light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients. Citation Format: Justin M. Drake, Evan O. Paull, Nicholas A. Graham, John K. Lee, Bryan A. Smith, Tanya Stoyanova, Claire M. Faltermeier, Daniel E. Carlin, Ajay Vashisht, Jiaoti Huang, James A. Wohlschlegel, Thomas G. Graeber, Owen N. Witte, Joshua M. Stuart. Patient-specific druggable networks in lethal prostate cancer through proteome-guided multi-omic integration. [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 3882.

A Surgical Perspective on Targeted Therapy of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide, is difficult to treat and highly lethal. Since HCC is predominantly diagnosed in patients with cirrhosis, treatment planning must consider both the severity of liver disease and tumor burden. To minimize the impact to the patient while treating the tumor, techniques have been developed to target HCC. Anatomical targeting by surgical resection or locoregional therapies is generally reserved for patients with preserved liver function and minimal to moderate tumor burden. Patients with decompensated cirrhosis and small tumors are optimal candidates for liver transplantation, which offers the best chance of long-term survival. Yet, only 20%–30% of patients have disease amenable to anatomical targeting. For the majority of patients with advanced HCC, chemotherapy is used to target the tumor biology. Despite these treatment options, the five-year survival of patients in the United States with HCC is only 16%. In this review we provide a comprehensive overview of current approaches to target HCC. We also discuss emerging diagnostic and prognostic biomarkers, novel therapeutic targets identified by recent genomic profiling studies, and potential applications of immunotherapy in the treatment of HCC.

Abstract 4985: Notch1 as a key mediator in promoting advanced castration-resistant prostate cancer

The first line of treatment for men with advanced prostate cancers is androgen deprivation therapy. However, the disease commonly relapses in its lethal metastatic form referred to as castration-resistant prostate cancer (CRPC). CRPC is the primary cause of prostate cancer specific mortality in men. Current therapies including chemotherapeutic agents improve median overall survival by only few months. The mechanisms that distinguish clinically localized indolent tumors from lethal CRPC are unclear. Here we demonstrate that ectopic expression of Notch1 promotes progression to poorly differentiated carcinoma when combined with pathways that are altered in advanced disease but are insufficient to drive aggressive prostate cancer alone. Notch1 driven tumors are resistant to androgen deprivation. Transcriptional profiling reveals that these tumors display features of epithelial to mesenchymal transition, a morphological change associated with tumor aggressiveness and metastasis. Our study provides the first functional evidence that Notch1 signaling axis is a key mediator in promoting advanced prostate cancer and may represent a new therapeutic target for the advanced disease. Note: This abstract was not presented at the meeting. Citation Format: Tanya Stoyanova, Claire Faltermeier, Bryan Smith, Andrew Goldstein, Xi Zhang, Justin Drake, John Lee, Sandra Orellana, Steven Blum, Donghui Cheng, Kenneth Pienta, Jiaoti Huang, Owen Witte. Notch1 as a key mediator in promoting advanced castration-resistant prostate cancer. [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 4985. doi:10.1158/1538-7445.AM2015-4985