Indications and Outcomes for Deferred Cytoreductive Nephrectomy Following Immune Checkpoint Inhibitor Combination Therapy: Can Systemic Therapy be Withdrawn in Patients with No Evidence of Disease?

Background: Upfront cytoreductive nephrectomy (CN) is no longer the standard of care for patients with metastastic renal cell carcinoma (mRCC) with intermediate or poor prognosis according to the International mRCC Database Consortium categories. Objective: To investigate indications for CN following first-line ipilimumab-nivolumab, and assess management and outcomes for patients achieving no evidence of disease (NED) after CN. Design setting and participants: This was a retrospective cohort study among 125 patients with synchronous mRCC who received ipilimumab-nivolumab treatment between March 2019 and June 2022 at four European centres. At one of the four centres, nivolumab was stopped following NED. Outcome measurements and statistical analysis: We measured complete response of metastases (mCR) according to Response Evaluation Criteria in Solid Tumours 1.1; near-complete response of mestastases (mnCR) was defined as a >80% reduction in cumulative metastatic volume. Treatment-free survival (TFS), disease-free survival (DFS), progression-free survival (PFS), and cancer-specific survival (CSS) were determined. Results and limitations: At median follow-up of 25 mo, 23/125 patients (18%) had undergone deferred CN. Of 26 patients (21%) with mCR or mnCR, 19 (73%) underwent CN to achieve NED, of whom 11 (58%) discontinued nivolumab, with median TFS of 21 mo. For patients who continued (n = 8, 42%) versus discontinued nivolumab following NED, 2-yr DFS was 83% versus 60% (p = 0.675) and 3-yr CSS was 100% versus 70% (p = 0.325). Four patients underwent CN because of a dissociated response of the primary tumour and were still alive at median follow-up of 5 mo. Conclusions: CN can result in NED, durable DFS, and substantial time off systemic therapy. More collaborative data are required to ascertain the benefits of treatment discontinuation versus oncologic safety. Patient summary: In our study using real-world data, 18% of patients treated with immunotherapy underwent deferred kidney surgery. The majority were free of disease after 3 years. Half of the patients who stopped immunotherapy after surgery have been off therapy for 21 months or longer. Larger studies are needed to investigate the effect of kidney surgery and discontinuation of immunotherapy on survival.

Novel genetically engineered mouse models for clear cell renal cell carcinoma.

Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.

NDUFA4L2 reduces mitochondrial respiration resulting in defective lysosomal trafficking in clear cell renal cell carcinoma.

In clear cell renal cell carcinoma (ccRCC), activation of hypoxic signaling induces NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) expression. Over 90% of ccRCCs exhibit overexpression of NDUFA4L2, which we previously showed contributes to ccRCC proliferation and survival. The function of NDUFA4L2 in ccRCC has not been fully elucidated. NDUFA4L2 was reported to reduce mitochondrial respiration via mitochondrial complex I inhibition. We found that NDUFA4L2 expression in human ccRCC cells increases the extracellular acidification rate, indicative of elevated glycolysis. Conversely, NDUFA4L2 expression in non-cancerous kidney epithelial cells decreases oxygen consumption rate while increasing extracellular acidification rate, suggesting that a Warburg-like effect is induced by NDUFA4L2 alone. We performed mass-spectrometry (MS)-based proteomics of NDUFA4L2 associated complexes. Comparing RCC4-P (parental) ccRCC cells with RCC4 in which NDUFA4L2 is knocked out by CRISPR-Cas9 (RCC4-KO-643), we identified 3,215 proteins enriched in the NDUFA4L2 immunoprecipitates. Among the top-ranking pathways were "Metabolic Reprogramming in Cancer" and "Glycolysis Activation in Cancer (Warburg Effect)." We also show that NDUFA4L2 enhances mitochondrial fragmentation, interacts with lysosomes, and increases mitochondrial-lysosomal associations, as assessed by high-resolution fluorescence microscopy and live cell imaging. We identified 161 lysosomal proteins, including Niemann-Pick Disease Type C Intracellular Cholesterol Transporters 1 and 2 (NPC1, NPC2), that are associated with NDUFA4L2 in RCC4-P cells. RCC4-P cells have larger and decreased numbers of lysosomes relative to RCC4 NDUFA4L2 knockout cells. These findings suggest that NDUFA4L2 regulates mitochondrial-lysosomal associations and potentially lysosomal size and abundance. Consequently, NDUFA4L2 may regulate not only mitochondrial, but also lysosomal functions in ccRCC.

Transcriptional and metabolic remodeling in clear cell renal cell carcinoma caused by ATF4 activation and the integrated stress response (ISR).

Research has shown extensive metabolic remodeling in clear cell renal cell carcinoma (ccRCC), with increased glutathione (GSH) levels. We hypothesized that activating transcription factor-4 (ATF4) and the integrated stress response (ISR) induce a metabolic shift, including increased GSH accumulation, and that Vitamin A deficiency (VAD), found in ccRCCs, can also activate ATF4 signaling in the kidney. To determine the role of ATF4, we used publicly available RNA sequencing (RNA-seq) data sets from The Cancer Genomics Atlas. Subsequently, we performed RNA-seq and liquid chromatography-mass spectrometry-based metabolomics analysis of the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early-stage ccRCC. To validate our findings, we generated RCC4 cell lines with ATF4 gene edits (ATF4-knockout [KO]) and subjected these cells to metabolic isotope tracing. Analysis of variance, the two-sided Student's t test, and gene set enrichment analysis were used (p < 0.05) to determine statistical significance. Here we show that most human ccRCC tumors exhibit activation of the transcription factor ATF4. Activation of ATF4 is concomitant with enrichment of the ATF4 gene set and elevated expression of ATF4 target genes ASNS, ALDH1L2, MTHFD2, DDIT3 (CHOP), DDIT4, TRIB3, EIF4EBP1, SLC7A11, and PPP1R15A (GADD34). Transcript profiling and metabolomics analyses show that activated hypoxia-inducible factor-1α (HIF1α) signaling in our TRACK ccRCC murine model also induces an ATF4-mediated ISR. Notably, both normoxic HIF1α signaling in TRACK kidneys and VAD in wild-type kidneys diminish amino acid levels, increase ASNS, TRIB3, and MTHFD2 messenger RNA levels, and increase levels of lipids and GSH. By metabolic isotope tracing in human RCC4 kidney cancer parental and ATF4 gene-edited (ATF4-KO) cell lines, we show that ATF4 increases GSH accumulation in part via activation of the mitochondrial one-carbon metabolism pathway. Our results demonstrate for the first time that activation of ATF4 enhances GSH accumulation, increases purine and pyrimidine biosynthesis, and contributes to transcriptional and metabolic remodeling in ccRCC. Moreover, constitutive HIF1α expressed only in murine kidney proximal tubules activates ATF4, leading to the metabolic changes associated with the ISR. Our data indicate that HIF1α can promote ccRCC via ATF4 activation. Moreover, lack of Vitamin A in the kidney recapitulates aspects of the ISR.

The genomic landscape of metastatic clear cell renal cell carcinoma after systemic therapy.

Primary clear cell renal cell carcinoma (ccRCC) has been previously characterized, but the genomic landscape of metastatic ccRCC is largely unexplored. Here, we performed whole exome sequencing (WES) in 68 samples from 44 patients with ccRCC, including 52 samples from a metastatic site. SETD2, PBRM1, APC and VHL were the most frequently mutated genes in the metastatic ccRCC cohort. RBM10 and FBXW7 were also among the 10 most frequently mutated genes in metastatic tissues. Recurrent somatic copy number variations (CNV) were observed at the previously identified regions 3p25, 9p21 and 14q25, but also at 6p21 (CDKN1A) and 13q14 (RB1). No statistically significant differences were found between samples from therapy-naïve and pretreated patients. Clonal evolution analyses with multiple samples from 13 patients suggested that early appearance of CNVs at 3p25, 9p21 and 14q25 may be associated with rapid clinical progression. Overall, the genomic landscapes of primary and metastatic ccRCC seem to share frequent CNVs at 3p25, 9p21 and 14q25. Future work will clarify the implication of RBM10 and FBXW7 mutations and 6p21 and 13q14 CNVs in metastatic ccRCC.

Kinase Inhibitor Treatment of Patients with Advanced Cancer Results in High Tumor Drug Concentrations and in Specific Alterations of the Tumor Phosphoproteome.

Identification of predictive biomarkers for targeted therapies requires information on drug exposure at the target site as well as its effect on the signaling context of a tumor. To obtain more insight in the clinical mechanism of action of protein kinase inhibitors (PKIs), we studied tumor drug concentrations of protein kinase inhibitors (PKIs) and their effect on the tyrosine-(pTyr)-phosphoproteome in patients with advanced cancer. Tumor biopsies were obtained from 31 patients with advanced cancer before and after 2 weeks of treatment with sorafenib (SOR), erlotinib (ERL), dasatinib (DAS), vemurafenib (VEM), sunitinib (SUN) or everolimus (EVE). Tumor concentrations were determined by LC-MS/MS. pTyr-phosphoproteomics was performed by pTyr-immunoprecipitation followed by LC-MS/MS. Median tumor concentrations were 2-10 µM for SOR, ERL, DAS, SUN, EVE and >1 mM for VEM. These were 2-178 × higher than median plasma concentrations. Unsupervised hierarchical clustering of pTyr-phosphopeptide intensities revealed patient-specific clustering of pre- and on-treatment profiles. Drug-specific alterations of peptide phosphorylation was demonstrated by marginal overlap of robustly up- and downregulated phosphopeptides. These findings demonstrate that tumor drug concentrations are higher than anticipated and result in drug specific alterations of the phosphoproteome. Further development of phosphoproteomics-based personalized medicine is warranted.

NCI 6896: a phase I trial of vorinostat (SAHA) and isotretinoin (13-cis retinoic acid) in the treatment of patients with advanced renal cell carcinoma.

Preclinical studies suggest that histone deacetylase (HDAC) inhibitors may restore tumor sensitivity to retinoids and have synergistic anti-tumor activity when combined. We performed a Phase I clinical trial to evaluate the safety and preliminary efficacy of combining the oral HDAC inhibitor vorinostat and isotretinoin in patients with advanced renal cell carcinoma (RCC). Vorinostat was administered at 300 mg orally twice daily in combination with escalating doses of isotretinoin for 3 consecutive days per week. A standard 3 + 3 dose escalation design was used. Dose limiting toxicities (DLT) were assess during the first cycle to determine the maximum tolerated dose (MTD). Fourteen patients enrolled on the trial of which 12 were evaluable for toxicity (6 cohort 1; 3 cohort 2; 3 cohort 3) and 11 for tumor response. One patient in cohort 1 experienced a DLT (grade 3 depression). Common grade 1-2 toxicities included fatigue and GI effects (nausea, diarrhea, anorexia). MTD was established as vorinostat 300 mg with isoretinoin 0.5 mg/kg twice daily 3 days per week. Best responses in evaluable patients included 1 partial response and 9 stable disease, lasting a median of 3.7 months (range 1.8-10.4 months). The combination of vorinostat and isotretinoin is safe, tolerable and associated with responses in patients with refractory metastatic RCC.

Combined Metabolomics and Genome-Wide Transcriptomics Analyses Show Multiple HIF1α-Induced Changes in Lipid Metabolism in Early Stage Clear Cell Renal Cell Carcinoma.

The accumulation of lipids is a hallmark of human clear cell renal cell carcinoma (ccRCC). Advanced ccRCC tumors frequently show increased lipid biosynthesis, but the regulation of lipid metabolism in early stage ccRCC tumors has not been studied. Here, we performed combined transcriptomics and metabolomics on a previously characterized transgenic mouse model (TRAnsgenic Cancer of the Kidney, TRACK) of early stage ccRCC. We found that in TRACK kidneys, HIF1α activation increases transcripts of lipid receptors (Cd36, ACVRL1), lipid storage genes (Hilpda and Fabp7), and intracellular levels of essential fatty acids, including linoleic acid and linolenic acid. Feeding the TRACK mice a high-fat diet enhances lipid accumulation in the kidneys. These results show that HIF1α increases the uptake and storage of dietary lipids in this early stage ccRCC model. By then analyzing early stage human ccRCC specimens, we found similar increases in CD36 transcripts and increases in linoleic and linolenic acid relative to normal kidney samples. CD36 mRNA levels decreased, while FASN transcript levels increased with increasing ccRCC tumor stage. These results suggest that an increase in the lipid biosynthesis pathway in advanced ccRCC tumors may compensate for a decreased capacity of these advanced ccRCCs to scavenge extracellular lipids.

Validation of risk factors for recurrence of renal cell carcinoma: Results from a large single-institution series.

PURPOSE: To validate prognostic factors and determine the impact of obesity, hypertension, smoking and diabetes mellitus (DM) on risk of recurrence after surgery in patients with localized renal cell carcinoma (RCC). MATERIALS AND METHODS: We performed a retrospective cohort study among patients that underwent partial or radical nephrectomy at Weill Cornell Medicine for RCC and collected preoperative information on RCC risk factors, as well as pathological data. Cases were reviewed for radiographic evidence of RCC recurrence. A Cox proportional-hazards model was developed to determine the contribution of RCC risk factors to recurrence risk. Disease-free survival and overall survival were analyzed using the Kaplan-Meier method and log-rank test. RESULTS: We identified 873 patients who underwent surgery for RCC between the years 2000-2015. In total 115 patients (13.2%) experienced a disease recurrence after a median follow up of 4.9 years. In multivariate analysis, increasing pathological T-stage (HR 1.429, 95% CI 1.265-1.614) and Nuclear grade (HR 2.376, 95% CI 1.734-3.255) were independently associated with RCC recurrence. In patients with T1-2 tumors, DM was identified as an additional independent risk factor for RCC recurrence (HR 2.744, 95% CI 1.343-5.605). Patients with DM had a significantly shorter median disease-free survival (1.5 years versus 2.6 years, p = 0.004), as well as median overall survival (4.1 years, versus 5.8 years, p<0.001). CONCLUSIONS: We validated high pathological T-stage and nuclear grade as independent risk factors for RCC recurrence following nephrectomy. DM is associated with an increased risk of recurrence among patients with early stage disease.

Association of Smoking and Death from Genitourinary Malignancies: Analysis of the National Longitudinal Mortality Study.

PURPOSE: We explored the association between tobacco use and genitourinary cancer specific survival in a contemporary, nationally representative sample of the United States civilian population. MATERIALS AND METHODS: A total of 493,282 participants in the National Longitudinal Mortality Study who provided detailed tobacco information from 1993 to 2005 were included in study. Our primary outcome was death from bladder, kidney or prostate cancer. Cause of death was determined from death certificates. Analyzed smoking parameters included smoking status at the time of the survey, age at the start of smoking and home smoking rules. Multivariable Cox regression models were used to assess associations of different smoking parameters with bladder, kidney and prostate cancer specific mortality. RESULTS: During a 5-year followup 5.6% of participants who had ever smoked died compared to 3.1% of those who had never smoked (p <0.0001). Of those who died of bladder, kidney and prostate cancer 62%, 58% and 62%, respectively, were ever smokers. On multivariable analysis ever smoking was associated with bladder and kidney cancer mortality (HR 1.92, 95% CI 1.25-2.97, and HR 1.54, 95% CI 1.01-2.34, respectively). Additionally, starting to smoke during teenage years and smoking at home were associated with bladder cancer specific mortality (HR 2.14, 95% CI 1.28-3.56 and HR 2.99, 95% CI 1.34-6.65) and kidney cancer specific mortality (HR 1.65, 95% CI 1.03-2.66 and HR 2.84, 95% CI 1.54-5.23, respectively). However, only everyday smoking was associated with an increased risk of prostate cancer mortality (HR 1.81, 95% CI 1.30-2.53). CONCLUSIONS: In a nationally representative study we confirmed the association between smoking intensity and mortality from genitourinary malignancies. Starting to smoke at a younger age and smoking at home conferred a significantly higher risk of death from bladder and kidney cancers.

Rscreenorm: normalization of CRISPR and siRNA screen data for more reproducible hit selection.

BACKGROUND: Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. RESULTS: We present "rscreenorm", a method that standardizes the functional data ranges between screens using assay controls, and subsequently performs a piecewise-linear normalization to make data distributions across all screens comparable. In simulation studies, rscreenorm reduces false positives. Using two multiple-cell lines siRNA screens, rscreenorm increased reproducibility between 27 and 62% for hits, and up to 5-fold for non-hits. Using publicly available CRISPR-Cas screen data, application of commonly used median centering yields merely 34% of overlapping hits, in contrast with rscreenorm yielding 84% of overlapping hits. Furthermore, rscreenorm yielded at most 8% discordant results, whilst median-centering yielded as much as 55%. CONCLUSIONS: Rscreenorm yields more consistent results and keeps false positive rates under control, improving reproducibility of genetic screens data analysis from multiple cell lines.

Selection of Protein Kinase Inhibitors Based on Tumor Tissue Kinase Activity Profiles in Patients with Refractory Solid Malignancies: An Interventional Molecular Profiling Study.

LESSONS LEARNED: Clinically applicable tools are needed for treatment selection and repurposing of available protein kinase inhibitors (PKIs) in patients with advanced solid tumors refractory to standard treatment.Using a tyrosine kinase peptide substrate microarray, observed inhibitory activity in vitro could not sufficiently predict clinical benefit of treatment with the selected PKI. BACKGROUND: This exploratory molecular profiling study determined the feasibility and benefit of the selection of protein kinase inhibitors (PKIs) based on kinase activity profiling in patients with refractory solid malignancies. METHODS: Adult patients with biopsy-accessible refractory solid tumors were eligible. Per patient, the inhibitory potency of sunitinib, dasatinib, erlotinib, sorafenib, everolimus, and lapatinib was determined in tumor lysates from fresh biopsies using a tyrosine kinase peptide substrate microarray. The most active PKI in this in vitro assay was selected for treatment. RESULTS: Thirteen patients were enrolled in the feasibility part and underwent tumor biopsy. Of 12 patients in whom kinase activity profiling was performed, 11 started treatment with a selected PKI: dasatinib in 8, sunitinib in 2, and erlotinib in 1 patient(s). Eight patients were evaluable for response. One patient had stable disease (SD) >4 months on sunitinib; one patient had SD at 6 weeks but progressive disease (PD) at 12 weeks. The remaining patients had PD after 6 weeks of treatment. CONCLUSION: Kinase inhibition profiles of multiple PKIs can be reliably determined using fresh tumor biopsies from patients with refractory solid tumors. However, the current in vitro microarray selection approach insufficiently predicted clinical benefit of PKI treatment in these patients.

Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection.

Mass spectrometry-based phosphoproteomics of cancer cell and tissue lysates provides insight in aberrantly activated signaling pathways and potential drug targets. For improved understanding of individual patient's tumor biology and to allow selection of tyrosine kinase inhibitors in individual patients, phosphoproteomics of small clinical samples should be feasible and reproducible. We aimed to scale down a pTyr-phosphopeptide enrichment protocol to biopsy-level protein input and assess reproducibility and applicability to tumor needle biopsies. To this end, phosphopeptide immunoprecipitation using anti-phosphotyrosine beads was performed using 10, 5 and 1mg protein input from lysates of colorectal cancer (CRC) cell line HCT116. Multiple needle biopsies from 7 human CRC resection specimens were analyzed at the 1mg-level. The total number of phosphopeptides captured and detected by LC-MS/MS ranged from 681 at 10mg input to 471 at 1mg HCT116 protein. ID-reproducibility ranged from 60.5% at 10mg to 43.9% at 1mg. Per 1mg-level biopsy sample, >200 phosphopeptides were identified with 57% ID-reproducibility between paired tumor biopsies. Unsupervised analysis clustered biopsies from individual patients together and revealed known and potential therapeutic targets. SIGNIFICANCE: This study demonstrates the feasibility of label-free pTyr-phosphoproteomics at the tumor biopsy level based on reproducible analyses using 1mg of protein input. The considerable number of identified phosphopeptides at this level is attributed to an effective down-scaled immuno-affinity protocol as well as to the application of ID propagation in the data processing and analysis steps. Unsupervised cluster analysis reveals patient-specific profiles. Together, these findings pave the way for clinical trials in which pTyr-phosphoproteomics will be performed on pre- and on-treatment biopsies. Such studies will improve our understanding of individual tumor biology and may enable future pTyr-phosphoproteomics-based personalized medicine.

Lactic Acidosis in Prostate Cancer: Consider the Warburg Effect.

Lactic acidosis is a commonly observed clinical condition that is associated with a poor prognosis, especially in malignancies. We describe a case of an 81-year-old patient who presented with symptoms of tachypnea and general discomfort. Arterial blood gas analysis showed a high anion gap acidosis with a lactate level of 9.5 mmol/L with respiratory compensation. CT scanning showed no signs of pulmonary embolism or other causes of impaired tissue oxygenation. Despite treatment with sodium bicarbonate, the patient developed an adrenalin-resistant cardiac arrest, most likely caused by the acidosis. Autopsy revealed Gleason score 5 + 5 metastatic prostate cancer as the most probable cause of the lactic acidosis. Next-generation sequencing indicated a nonsense mutation in the TP53 gene (887delA) and an activating mutation in the PIK3CA gene (1634A>G) as candidate molecular drivers. This case demonstrates the prevalence and clinical relevance of metabolic reprogramming, frequently referred to as "the Warburg effect," in patients with prostate cancer.

Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer.

Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5-10 µg of lysate protein and up to 400 µM adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5-7 µg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides.

Novel drugs that target the metabolic reprogramming in renal cell cancer.

Molecular profiling studies of tumor tissue from patients with clear cell renal cell cancer (ccRCC) have revealed extensive metabolic reprogramming in this disease. Associations were found between metabolic reprogramming, histopathologic Fuhrman grade, and overall survival of patients. Large-scale genomics, proteomics, and metabolomic analyses have been performed to identify the molecular players in this process. Genes involved in glycolysis, the pentose phosphate pathway, glutamine metabolism, and lipogenesis were found to be upregulated in renal cell cancer (RCC) specimens as compared to normal tissue. Preclinical research indicates that mutations in VHL, FBP1, and the PI3K-AKT-mTOR pathway drives aerobic glycolysis through transcriptional activation of the hypoxia-inducible factors (HIF). Mechanistic studies revealed glutamine as an important source for de novo fatty acid synthesis through reductive carboxylation. Amplification of MYC drives reductive carboxylation. In this review, we present a detailed overview of the metabolic changes in RCC in conjunction with potential novel therapeutics. We discuss preclinical studies that have investigated targeted agents that interfere with various aspects of tumor cell metabolism and emphasize their impact specifically on glycolysis, lipogenesis, and tumor growth. Furthermore, we describe a number of phase 1 and 2 clinical trials that have been conducted with these agents.

Evaluation of potential circulating biomarkers for prediction of response to chemoradiation in patients with glioblastoma.

Surgery followed by chemoradiation and adjuvant chemotherapy is standard of care for patients with a glioblastoma (GBM). Due to its limited benefit, an upfront method to predict dismal outcome would prevent unnecessary toxic treatment. We searched for a predictive blood derived biomarker in a cohort of 55 patients with GBM. Increasing age (HR 1.03, 95 % CI 1.01-1.06), and postoperative tumor residue (HR 1.07, 95 % CI 1.02-1.15) were independently associated with unfavourable progression free survival (PFS) in these patients. Corticosteroid use before start of chemoradiaton was strongly predictive for outcome (HR 3.26, 95 % CI 1.67-6.39) with a mean PFS and OS in patients using corticosteroids of 7.3 and 14.6 months, versus 16.1 and 21.6 months in patients not using corticosteroids (p = 0.0005, p < 0.0067 respectively). Despite earlier reports, blood concentrations of YKL-40, Fetuin-a and haptoglobin were not predictive for response. In addition, serum peptide profiles, determined by MALDI-TOF mass spectroscopy, were not predictive as well. In conclusion, further biomarker discovery studies are needed to predict treatment outcome for patients with GBM in the near future.

Sunitinib activates Axl signaling in renal cell cancer.

Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.

Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics.

BACKGROUND: Mass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of tyrosine phosphorylation on proteins and tyrosine kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free phosphotyrosine-based phosphoproteomics. METHODS: Phosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed using agarose bead-coupled phosphotyrosine antibodies P-Tyr-1000 (N=3) and 4G10 (N=3). NanoLC-MS/MS was performed using a Q Exactive mass spectrometer. For relative quantitation of protein phosphorylation, spectral counts of phosphoproteins and ion intensities of phosphopeptides were determined using MaxQuant. RESULTS: From the 3 samples incubated with P-Tyr-1000 a total of 689 phosphopeptides were identified with 60% ID reproducibility. The phosphopeptide capture using 4G10 resulted in a total of 421 at 46% ID reproducibility. The P-Tyr-1000 was applied to EGFR mutated U87 cells. Erlotinib reduced EGFR phosphorylation with 59% at y978, y1125, y1138, y1172, and y1197. EGFR inhibition was accompanied by enhanced phosphorylation of FYN, MET, PTK2, DYRK1A, MAPK1 and EPHA2. CONCLUSION: The P-Tyr-1000 phosphotyrosine antibody performs superiorly when compared to 4G10 antibody for label-free phosphotyrosine-based phosphoproteomics. This workflow allows evaluation of drug target phosphorylation and may give insights in the pharmacodynamic effects of tyrosine kinase inhibitors. CLINICAL SIGNIFICANCE: In the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomic analyses have meanwhile emerged as a sophisticated approach for the determination of protein phosphorylation. These analyses rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than 4G10 antibody, while including almost all 4G10 captured phosphopeptides. The workflow can be reproducibly performed at intermediate protein input levels of 10mg. Furthermore, application of the P-Tyr-1000 antibody in a standardized phosphoproteomics workflow allows relative quantitation of drug target inhibition and provides insights in alternative signaling pathways in cancer cells. This article is part of a Special Issue entitled: HUPO 2014.