A Bivalent Inhibitor of Prostate Specific Membrane Antigen Radiolabeled with Copper-64 with High Tumor Uptake and Retention.

Molecules containing lysine-ureido-glutamate functional groups bind to the active site of prostate specific membrane antigen, which is overexpressed in prostate cancer. To prepare copper radiopharmaceuticals for the diagnosis and therapy of prostate cancer, macrobicyclic sarcophagine ligands tethered to either one or two lysine-ureido-glutamate functional groups through an appropriate linker have been prepared. Sarcophagine ligands can be readily radiolabeled with positron-emitting copper-64 at room temperature. The bivalent agent, in which two targeting groups are tethered to a single copper complex, dramatically outperforms the monomeric agent with respect to tumor uptake and retention. The high tumor uptake, low background, and prolonged tumor retention, even at 24 hours post injection, suggest the bivalent agent is a promising diagnostic for prostate cancer and could be used for prospective dosimetry for therapy with a copper-67 variant.

Palbociclib synergizes with BRAF and MEK inhibitors in treatment naïve melanoma but not after the development of BRAF inhibitor resistance.

Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.

Long term, continuous exposure to panobinostat induces terminal differentiation and long term survival in the TH-MYCN neuroblastoma mouse model.

Neuroblastoma is the most common extra-cranial malignancy in childhood and accounts for ∼15% of childhood cancer deaths. Amplification of MYCN in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N-Myc and regulate numerous cellular processes via epigenetic modulation, including differentiation. In this study, we used the TH-MYCN mouse model of neuroblastoma to investigate the antitumor activity of the pan-HDAC inhibitor, panobinostat. In particular we sought to explore the impact of long term, continuous panobinostat exposure on the epigenetically driven differentiation process. Continuous treatment of tumor bearing TH-MYCN transgenic mice with panobinostat for nine weeks led to a significant improvement in survival as compared with mice treated with panobinostat for a three-week period. Panobinostat induced rapid tumor regression with no regrowth observed following a nine-week treatment period. Initial tumor response was associated with apoptosis mediated via upregulation of BMF and BIM. The process of terminal differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N-Myc expression, occurred following prolonged exposure to the drug. RNA-sequencing analysis of tumors from treated animals confirmed significant upregulation of gene pathways associated with apoptosis and differentiation. Together our data demonstrate the potential of panobinostat as a novel therapeutic strategy for high-risk neuroblastoma patients.

The genomic landscape of phaeochromocytoma.

Phaeochromocytomas (PCCs) and paragangliomas (PGLs) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PCC/PGL cases are associated with heritable syndromes involving 13 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PCC/PGL, we applied exome sequencing, high-density SNP-array analysis, and RNA sequencing to 36 PCCs and four functional PGL tumours. All tumours displayed low mutation frequency, in contrast to frequent large segmental copy-number alterations, aneuploidy, and evidence for chromothripsis in one case. Multi-region sampling of one benign familial PCC tumour provided evidence for the timing of mutations during tumourigenesis and ongoing clonal evolution. Thirty-one of 40 (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PCC/PGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation. A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function. We revisited the gene-expression subtyping of PCC/PGL by integrating published microarray data with our RNA-seq data, enabling the identification of six robust gene-expression subtypes. The majority of cases in our cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PCC/PGL. Our data suggest there are yet unknown PCC/PGL cancer genes that can phenocopy MAX mutant PCC/PGL tumours. This study provides new insight into the molecular diversity and genetic origins of PCC/PGL tumours.

Peptide receptor chemoradionuclide therapy in small cell carcinoma: from bench to bedside.

PURPOSE: Small cell cancers (SmCC), whether pulmonary (SCLC) or extrapulmonary, have a poor prognosis unless localised at diagnosis. Given a proportion of these cancers express somatostatin receptor subtype 2 (SSTR2), we aimed to investigate the efficacy of targeted peptide receptor chemoradionuclide therapy (PRCRT). METHODS: In this preclinical study, we used a SCLC xenograft mouse model with high expression of SSTR2 to investigate the effect of peptide receptor radionuclide therapy (PRRT) with chemotherapy compared to either alone. We subsequently explored the clinical utility in a patient with SmCC with high SSTR expression treated with PRCRT. RESULTS: Robust expression of SSTR2 in NCI-H69 SCLC xenografts was documented by (68)Ga-DOTA-octreotate (GaTate) (tumour to background uptake ratio = 35). The combination of PRRT using (177)Lu-DOTA-octreotate (LuTate) with carboplatin/etoposide (C/E) chemotherapy was more effective than either LuTate or C/E alone for regression of the NCI-H69 model (p value < 0.05). PRCRT was associated with significantly prolonged survival versus PRRT (p value = 0.0001) or chemotherapy alone (p value = 0.0058). In the subsequent case study, a patient with relapsed SmCC with high SSTR2 expression on GaTate PET underwent PRCRT with radiosensitising etoposide with evidence of a complete metabolic response for 4 months. CONCLUSION: Given the limited treatment options in this setting, PRCRT is a promising therapeutic option for SSTR2-expressing SmCC.

A genome-wide CRISPR/Cas9 screen identifies DNA-PK as a sensitiser to 177Lutetium-DOTA-octreotate radionuclide therapy.

Peptide receptor radionuclide therapy (PRRT) using 177Lutetium-DOTA-octreotate (LuTate) for neuroendocrine tumours (NET) is now an approved treatment available in many countries, though primary or secondary resistance continue to limit its effectiveness or durability. We hypothesised that a genome-wide CRISPR/Cas9 screen would identify key mediators of response to LuTate and gene targets that might offer opportunities for novel combination therapies for NET patients. Methods: We utilised a genome-wide CRISPR-Cas9 screen in LuTate-treated cells to identify genes that impact on the sensitivity or resistance of cells to LuTate. Hits were validated through single-gene knockout. LuTate-resistant cells were assessed to confirm LuTate uptake and retention, and persistence of somatostatin receptor 2 (SSTR2) expression. Gene knockouts conferring LuTate sensitivity were further characterised by pharmacological sensitisation using specific inhibitors and in vivo analysis of the efficacy of these inhibitors in combination with LuTate. Results: The CRISPR-Cas9 screen identified several potential targets for both resistance and sensitivity to PRRT. Two gene knockouts which conferred LuTate resistance in vitro, ARRB2 and MVP, have potential mechanisms related to LuTate binding and retention, and modulation of DNA-damage repair (DDR) pathways, respectively. The screen showed that sensitivity to LuTate treatment in vitro can be conferred by the loss of a variety of genes involved in DDR pathways, with loss of genes involved in Non-Homologous End-Joining (NHEJ) being the most lethal. Loss of the key NHEJ gene, PRKDC (DNA-PK), either by gene loss or inhibition by two different inhibitors, resulted in significantly reduced cell survival upon exposure of cells to LuTate. In SSTR2-positive xenograft-bearing mice, the combination of nedisertib (a DNA-PK specific inhibitor) and LuTate produced a more robust control of tumour growth and increased survival compared to LuTate alone. Conclusions: DDR pathways are critical for sensing and repairing radiation-induced DNA damage, and our study shows that regulation of DDR pathways may be involved in both resistance and sensitivity to PRRT. Additionally, the use of a DNA-PK inhibitor in combination with LuTate PRRT significantly improves the efficacy of the treatment in pre-clinical models, providing further evidence for the clinical efficacy of this combination.

Preclinical characterization of 18F-D-FPHCys, a new amino acid-based PET tracer.

PURPOSE: The imaging potential of a new (18)F-labelled methionine derivative, S-(3-[(18)F]fluoropropyl)-D-homocysteine ((18)F-D-FPHCys), and its selectivity for amino acid transporter subtypes were investigated in vitro and by imaging of human tumour xenografts. METHODS: Expression of members of the system L (LAT isoforms 1-4 and 4F2hc) and ASCT (ASCT isoforms 1 and 2) amino acid transporter subclasses were assessed by quantitative real-time PCR in four human tumour models, including A431 squamous cell carcinoma, PC3 prostate cancer, and Colo 205 and HT-29 colorectal cancer lines. The first investigations for the characterization of (18)F-D-FPHCys were in vitro uptake studies by comparing it with [1-(14)C]-L-methionine ((14)C-MET) and in vivo by PET imaging. In addition, the specific involvement of LAT1 transporters in (18)F-D-FPHCys accumulation was tested by silencing LAT1 mRNA transcription with siRNAs. To determine the proliferative activity in tumour xenografts ex vivo, Ki-67 staining was used as a biomarker. RESULTS: A431 cells showed the highest (18)F-D-FPHCys uptake in vitro and in vivo followed by Colo 205, PC3 and HT-29. A similar pattern of retention was observed with (14)C-MET. (18)F-D-FPHCys retention was strongly correlated with LAT1 expression both in vitro (R(2) = 0.85) and in vivo (R(2) = 0.99). Downregulation of LAT1 by siRNA inhibited (18)F-D-FPHCys uptake, demonstrating a clear dependence on this transporter for tumour uptake. Furthermore, (18)F-D-FPHCys accumulation mirrored cellular proliferation. CONCLUSION: The favourable properties of (18)F-D-FPHCys make this tracer a promising imaging probe for detection of tumours as well as for the noninvasive evaluation and monitoring of tumour growth.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Enhancing the anti-tumour activity of 177Lu-DOTA-octreotate radionuclide therapy in somatostatin receptor-2 expressing tumour models by targeting PARP.

Peptide receptor radionuclide therapy (PRRT) is an important treatment option for patients with somatostatin receptor-2 (SSTR2)-expressing neuroendocrine tumour (NET) though tumour regression occurs in only a minority of patients. Therefore, novel PRRT regimens with improved therapeutic activity are needed. Radiation induced DNA damage repair is an attractive therapeutic target to increase PRRT efficacy and consequently, we have characterised a panel of preclinical models for their SSTR2 expression, in vivo growth properties and response to 177Lu-DOTA-octreotate (LuTate) PRRT to identify models with features suitable for evaluating novel therapeutic combinations. In vitro studies using the SSTR2 expressing AR42J model demonstrate that the combination of LuTate and the small molecule Poly(ADP-ribose) polymerase-1 (PARP) inhibitor, talazoparib led to increased DNA double strand breaks, as assessed by γ-H2AX foci formation, as compared to LuTate alone. Furthermore, using the AR42J tumour model in vivo we demonstrate that the combination of LuTate and talazoparib significantly improved the anti-tumour efficacy of LuTate alone. These findings support the clinical evaluation of the combination of LuTate and PARP inhibition in SSTR2-expressing NET.

Bivalent Inhibitors of Prostate-Specific Membrane Antigen Conjugated to Desferrioxamine B Squaramide Labeled with Zirconium-89 or Gallium-68 for Diagnostic Imaging of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a carboxypeptidase that is overexpressed in prostate cancer and is an excellent candidate for targeted diagnostic imaging and therapy. Lysine-ureido-glutamate inhibitors of PSMA radiolabeled with positron-emitting radionuclides can be used for diagnostic imaging with positron emission tomography (PET). A squaramide ester derivative of desferrioxamine B (H3DFOSq) was used to prepare four new agents with either one or two lysine-ureido-glutamate pharmacophores. The H3DFOSq ligand can be used to form stable complexes with either of the positron-emitting radionuclides gallium-68 (t1/2 = 68 min) or zirconium-89 (t1/2 = 3.3 days). The complexes were evaluated in PSMA-positive xenograft mouse models. Bivalent inhibitors, where two pharmacophores are tethered to a single DFOSq ligand, have better tumor uptake than their monovalent analogues. The ligands presented here, which can be labeled with either gallium-68 or zirconium-89, have the potential to increase the number of clinical sites that can perform diagnostic PET imaging.

The heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line.

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor kappaB ligand (RANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence caused by 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

Rapid kit-based (68)Ga-labelling and PET imaging with THP-Tyr(3)-octreotate: a preliminary comparison with DOTA-Tyr(3)-octreotate.

BACKGROUND: Ge/(68)Ga generators provide an inexpensive source of a PET isotope to hospitals without cyclotron facilities. The development of new (68)Ga-based molecular imaging agents and subsequent clinical translation would be greatly facilitated by simplification of radiochemical syntheses. We report the properties of a tris(hydroxypyridinone) conjugate of the SSTR2-targeted peptide, Tyr(3)-octreotate (TATE), and compare the (68)Ga-labelling and biodistribution of [(68)Ga(THP-TATE)] with the clinical radiopharmaceutical [(68)Ga(DOTATATE)]. METHODS: A tris(hydroxypyridinone) with a pendant isothiocyanate group was conjugated to the primary amine terminus of H2N-PEG2-Lys(iv-Dde)(5)-TATE, and the resulting conjugate was deprotected to provide THP-TATE. THP-TATE was radiolabelled with (68)Ga(3+) from a (68)Ge/(68)Ga generator. In vitro uptake was assessed in SSTR2-positive 427-7 cells and SSTR2-negative 427 (parental) cells. Biodistribution of [(68)Ga(THP-TATE)] was compared with that of [(68)Ga(DOTATATE)] in Balb/c nude mice bearing SSTR2-positive AR42J tumours. PET scans were obtained 1 h post-injection, after which animals were euthanised and tissues/organs harvested and counted. RESULTS: [(68)Ga(THP-TATE)] was radiolabelled and formulated rapidly in <2 min, in ≥95 % radiochemical yield at pH 5-6.5 and specific activities of 60-80 MBq nmol(-1) at ambient temperature. [(68)Ga(THP-TATE)] was rapidly internalised into SSTR2-positive cells, but not SSTR2-negative cells, and receptor binding and internalisation were specific. Animals administered [(68)Ga(THP-TATE)] demonstrated comparable SSTR2-positive tumour activity (11.5 ± 0.6 %ID g(-1)) compared to animals administered [(68)Ga(DOTATATE)] (14.4 ± 0.8 %ID g(-1)). Co-administration of unconjugated Tyr(3)-octreotate effectively blocked tumour accumulation of [(68)Ga(THP-TATE)] (2.7 ± 0.6 %ID g(-1)). Blood clearance of [(68)Ga(THP-TATE)] was rapid and excretion was predominantly renal, although compared to [(68)Ga(DOTATATE)], [(68)Ga(THP-TATE)] exhibited comparatively longer kidney retention. CONCLUSIONS: Radiochemical synthesis of [(68)Ga(THP-TATE)] is significantly faster, proceeds under milder conditions, and requires less manipulation than that of [(68)Ga(DOTATATE)]. A (68)Ga-labelled tris(hydroxypyridinone) conjugate of Tyr(3)-octreotate demonstrates specificity and targeting affinity for SSTR2 receptors, with comparable in vivo targeting affinity to the clinical PET tracer, [(68)Ga(DOTATATE)]. Thus, peptide conjugates based on tris(hydroxypyridinones) are conducive to translation to kit-based preparation of PET tracers, enabling the expansion and adoption of (68)Ga PET in hospitals and imaging centres without the need for costly automated synthesis modules.

UV-Associated Mutations Underlie the Etiology of MCV-Negative Merkel Cell Carcinomas.

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.

Preclinical FLT-PET and FDG-PET imaging of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901.

INTRODUCTION: The Aurora kinases play a key role in mitosis and have recently been identified as attractive targets for therapeutic intervention in cancer. The aim of this study was therefore to investigate the utility of 3'-[(18)F]fluoro-3'-deoxythymidine (FLT) and 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) for assessment of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901. METHODS: Balb/c nude mice bearing HCT116 colorectal xenografts were treated with up to 30mg/kg TAK 901 or vehicle intravenously twice daily for two days on a weekly cycle. Tumor growth was monitored by calliper measurements and PET imaging was performed at baseline, day 4, 8, 11 and 15. Tumors were harvested at time points corresponding to days of PET imaging for analysis of ex vivo markers of cell proliferation and metabolism together with markers of Aurora B kinase inhibition including phospho-histone H3 (pHH3) and senescence associated ß-galactosidase. RESULTS: Tumor growth was inhibited by 60% on day 12 of 30mg/kg TAK-901 therapy. FLT uptake was significantly reduced by day 4 of treatment and this corresponded with reduction in bromodeoxyuridine and pHH3 staining by immunohistochemistry. All biomarkers rebounded towards baseline levels by the commencement of the next treatment cycle, consistent with release of Aurora B kinase suppression. TAK-901 therapy had no impact on glucose metabolism as assessed by FDG uptake and GLUT1 staining by immunohistochemistry. CONCLUSIONS: FLT-PET, but not FDG-PET, is a robust non-invasive imaging biomarker of early HCT116 tumor response to the on-target effects of the multi-targeted Aurora B kinase inhibitor, TAK-901. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This is the first report to demonstrate the impact of the multi-targeted Aurora B kinase inhibitor, TAK-901 on tumor FLT uptake. The findings provide a strong rationale for the evaluation of FLT-PET as an early biomarker of tumor response in the early phase clinical development of this compound.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

The mTORC1 inhibitor everolimus prevents and treats Eµ-Myc lymphoma by restoring oncogene-induced senescence.

UNLABELLED: MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in Eµ-Myc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of Eµ-Myc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established Eµ-Myc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes. SIGNIFICANCE: This work provides novel insights into the requirements for MYC-induced oncogenesis by showing that mTORC1 activity is necessary to bypass senescence during transformation of B lymphocytes. Furthermore, tumor eradication through senescence elicited by targeted inhibition of mTORC1 identifies a previously uncharacterized mechanism responsible for significant anticancer activity of rapamycin analogues and serves as proof-of-concept that senescence can be harnessed for therapeutic benefit

Relationship between epidermal growth factor receptor status, p16(INK4A), and outcome in head and neck squamous cell carcinoma.

BACKGROUND: Human papilloma virus (HPV) infection is a powerful prognostic biomarker in head and neck squamous cell carcinoma (HNSCC). Increased epidermal growth factor receptor (EGFR) gene copy number and protein expression have been reported to be negative predictors of outcome. This study examined the relationship between HPV status, EGFR gene copy number, EGFR protein expression, and clinical outcome in HNSCC patients treated with chemoradiation. METHODS: HPV status was determined using p16(INK4A) immunohistochemistry (IHC), EGFR gene copy number was evaluated with FISH, and EGFR protein expression by IHC in 212 subjects. RESULTS: EGFR FISH was positive in 41 of 204 (20%) patients and was negatively correlated with failure-free survival (FFS; HR = 1.84, P = 0.027) and overall survival (OS; HR = 1.78, P = 0.082). For p16(INK4A), 85 of 200 (42.5%) patients were found to be p16 positive, including 75 of 131 (57%) with oropharyngeal cancer. Patients with p16-positive oropharyngeal cancer had significantly improved FFS (HR = 0.28, P < 0.001) and OS (HR = 0.31, P = 0.002). Only 2 of 126 (1.6%) oropharyngeal cancer patients were found to be p16+/EGFR FISH+. EGFR IHC was positive in 81 of 93 (87%) of patients and was associated with poorer FFS (HR = 1.98, P = 0.35) and OS (HR = 2.52, P = 0.22). CONCLUSIONS: Increased EGFR gene copy number is largely restricted to p16(INK4A)-negative oropharyngeal cancer. Although p16(INK4A) and EGFR FISH are both predictive of outcome in univariate analyses, only p16(INK4A) remains independently predictive. IMPACT: Knowledge of HPV and EGFR status can have implications for treatment options and prognosis in HNSCC.

18F-FLT PET as a surrogate marker of drug efficacy during mTOR inhibition by everolimus in a preclinical cisplatin-resistant ovarian tumor model.

UNLABELLED: Targeting the mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming cisplatin resistance in ovarian cancer patients. Because mTOR inhibition affects cell proliferation, we aimed to study whether 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET could be useful for monitoring early response to treatment with mTOR inhibitors in an animal model of cisplatin-resistant ovarian tumor. METHODS: BALB/c nude mice bearing subcutaneous human SKOV3 ovarian cancer xenografts were treated with either the mTOR inhibitor everolimus (5 mg/kg) or vehicle, and (18)F-FLT PET was performed at baseline, day 2, and day 7 of treatment. (18)F-FLT uptake was evaluated by calculation of mean standardized uptake value (SUVmean) corrected for partial-volume effect. Ex vivo immunohistochemistry studies were performed on separate cohorts of mice treated as above and sacrificed at the same time points as for the PET studies. The ex vivo analysis included bromodeoxyuridine incorporation as a marker of cell proliferation, and phosphorylation of ribosomal protein S6 as a downstream marker of mTOR activation. RESULTS: During the treatment period, no significant change in tumor (18)F-FLT uptake was observed in the vehicle group, whereas in everolimus-treated mice, (18)F-FLT SUVmean decreased by 33% (P = 0.003) at day 2 and 66% (P < 0.001) at day 7, compared with baseline. Notably, the reduction of (18)F-FLT uptake observed at day 2 in the everolimus group preceded changes in tumor volume, and a significant difference in (18)F-FLT uptake was observed between vehicle and drug-treated tumors at both day 2 (P = 0.0008) and day 7 (P = 0.01). In ex vivo studies, everolimus treatment resulted in a 98% reduction in phosphorylated ribosomal protein S6 immunostaining at day 2 (P = 0.02) and 91% reduction at day 7 (P = 0.003), compared with the vehicle group. Bromodeoxyuridine incorporation was reduced by 65% at day 2 (not significant) and by 41% at day 7 (P = 0.02) in drug versus vehicle groups. CONCLUSION: Reduction in (18)F-FLT uptake correlates well with the level of mTOR inhibition by everolimus in the SKOV3 ovarian tumor model. These data suggest that early treatment monitoring by (18)F-FLT PET may be of use in future preclinical or clinical trials evaluating treatment of cisplatin-resistant ovarian tumors by mTOR inhibitors.