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Supplementary Data from Preclinical Development of the Class-I–Selective Histone Deacetylase Inhibitor OKI-179 for the Treatment of Solid Tumors

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I–selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies.

Supplementary Figure from Preclinical Development of the Class-I–Selective Histone Deacetylase Inhibitor OKI-179 for the Treatment of Solid Tumors

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I–selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies.

To evaluate the relative rates of malignancy in women with single and multiple polyps presenting to a UK Cancer Centre with postmenopausal bleeding (PMB).A retrospective review of patients treated at Royal Derby Hospital (RDH) for PMB who underwent outpatient hysteroscopy based on ultrasonographic suspicion of endometrial polyps between May 2014 to December 2019. The main outcome measure was the rates of precancerous and malignant histology for single or multiple polyps. The secondary outcomes assessed the influence of risk factors on the rates of malignancy within the single and multiple polyps groups.The study population was 851 women of which 533 were in the single polyp group and 318 in the multiple polyps group. The multiple polyps group (mean age 65.2 years) was older compared to the single polyp group (mean age 62.1 years), P = 0.0001. Elevated rates of cancer was driven most significantly by endometrioid cancer in the multiple polyps compared to single polyp group, with rates of 50/314 (16 %) and 28/512 (5.5 %) respectively, P=0.00001. For rarer histologies there was no significant difference between the proportion of serous, carcinosarcomas and clear cell cancers between those with single compared to multiple polyps (P 0.05). Significantly more endometrial hyperplasia with atypia (AEH) was found in the multiple polyps compared to single polyp group, with rates of 18/314 (5.7 %) and 15/512 (2.9 %) respectively, P = 0.046.Our study found increased rates of endometrioid cancer and its precursor, AEH within the multiple polyps compared to the single polyps groups. Future risk predicting algorithms should consider incorporating single and multiple polyps as part of their risk model.

Targeted, catalytic degradation of oncoproteins using heterobifunctional small molecules is an attractive modality, particularly for hematologic malignancies, which are often initiated by aberrant transcription factors and are challenging to drug with inhibitors. BRD4, a member of the bromodomain and extraterminal family, is a core transcriptional and epigenetic regulator that recruits the P-TEFb complex, which includes Cdk9 and cyclin T, to RNA polymerase II (pol II). Together, BRD4 and CDK9 phosphorylate serine 2 (pSer2) of heptad repeats in the C-terminal domain of RPB1, the large subunit of pol II, promote transcriptional elongation. Small-molecule degraders of BRD4 have shown encouraging efficacy in preclinical models for several tumor types but less efficacy in other cancers including small-cell lung cancer (SCLC) and pancreatic cancer. Here, we evaluated CFT-2718, a new BRD4-targeting degrader with enhanced catalytic activity and in vivo properties. In vivo, CFT-2718 has significantly greater efficacy than the CDK9 inhibitor dinaciclib in reducing growth of the LX-36 SCLC patient-derived xenograft (PDX) model and performed comparably to dinaciclib in limiting growth of the PNX-001 pancreatic PDX model. In vitro, CFT-2718 reduced cell viability in four SCLC and two pancreatic cancer models. In SCLC models, this activity significantly exceeded that of dinaciclib; furthermore, CFT-2718 selectively increased the expression of cleaved PARP, an indicator of apoptosis. CFT-2718 caused rapid BRD4 degradation and reduced levels of total and pSer2 RPB1 protein. These and other findings suggest that BRD-mediated transcriptional suppression merits further exploration in the setting of SCLC.

The BRAF kinase plays a critical role in the MAPK signaling pathway and is mutated in ~8% of all human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), which is found in >70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because nearly all patients progress, due to either primary or acquired resistance. A bifunctional degradation activating compound (or BiDACTM degrader) may address the liabilities of approved drugs by overcoming, or preventing the emergence of, resistance to BRAF inhibitors. Here we describe CFT1946, an orally bioavailable cereblon-based BiDAC degrader of BRAF V600 mutant proteins, and provide an overview of the medicinal chemistry path leading to its discovery. CFT1946 degrades BRAF V600 mutant proteins, while maintaining exquisite selectivity against the proteome, sparing wild type BRAF (BRAF-WT), ARAF, and CRAF. In A375 cells, CFT1946 potently degraded BRAF V600E and inhibited ERK phosphorylation and cell growth while having no effect in the mutant KRAS, BRAF-WT driven cell line HCT116. In the A375 xenograft model, oral delivery of CFT1946 at 10 mg/kg PO BID resulted in deeper and more durable tumor regression compared to a clinically relevant dose of encorafenib. Further evaluation of CFT1946 in an engineered, clinically relevant BRAFi-resistant A375 cell line (endogenous BRAF V600E + engineered expression of NRAS Q61K) demonstrated that CFT1946 both degraded BRAF V600E and caused a loss of viability in these cells, while treatment with encorafenib had no effect. In xenografts derived from this BRAFi-resistant cell line, oral dosing of CFT1946 as a single agent led to tumor growth inhibition, while treatment with a clinically relevant dose of encorafenib had no effect on tumor growth. Furthermore, dosing CFT1946 in combination with the MEK inhibitor, trametinib, resulted in significant tumor regression, whereas combining encorafenib with the same dose of trametinib had no effect. The medicinal chemistry campaign resulting in CFT1946 focused on the improvement of in vivo pharmacokinetics and rational linker design to achieve high oral bioavailability in a beyond Rule of 5 heterobifunctional degrader. The preclinical data presented herein support the planned Phase 1/2 clinical trial of CFT1946 for the treatment of BRAF-V600 mutant solid tumors. Citation Format: Yanke Liang, Mathew E. Sowa, Katrina L. Jackson, Jeffrey R. Simard, Bridget Kreger, Ping Li, Laura Poling, Joelle Baddour, Andrew Good, Hongwei Huang, Scott Eron, Christopher G. Nasveschuk, Robert Yu, Mark Fitzgerald, Victoria Garza, Morgan W. O’Shea, Gesine Veits, Jeremy Y. Yap, Moses Moustakim, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe S. Patel, Richard Deibler, Kyle S. Cole, David Cocozziello, Fazlur Rahman, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Roy M. Pollock, Stewart L. Fisher. The discovery and characterization of CFT1946: A potent, selective, and orally bioavailable degrader of mutant BRAF for the treatment of BRAF-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3425.

Recent advances in targeted treatment for cholangiocarcinoma have focused on fibroblast growth factor (FGF) signaling. There are four receptor tyrosine kinases that respond to FGFs, and posttranslational processing has been demonstrated for each FGF receptor. Here, we investigated the role of N-linked glycosylation on the processing and function of FGFR4. We altered glycosylation through enzymatic deglycosylation, small molecule inhibition of glycosyltransferases, or through site-directed mutagenesis of selected asparagine residues in FGFR4. Signaling was tested through caspase activation, migration, and subcellular localization of FGFR4. Our data demonstrate that FGFR4 has multiple glycoforms, with predominant bands relating to the full-length receptor that has a high mannose- or hybrid-type form and a complex-type glycan form. We further identified a set of faster migrating FGFR4 bands that correspond to the intracellular kinase domain, termed FGFR4 intracellular domain (R4-ICD). These glycoforms and R4-ICD were detected in human cholangiocarcinoma tumor samples, where R4-ICD was predominant. Removal of glycans in intact cells by enzymatic deglycosylation resulted in increased processing to R4-ICD. Inhibition of glycosylation using NGI-1, an oligosaccharyltransferase inhibitor, reduced both high mannose- or hybrid- and complex-type glycan forms of FGFR4, increased processing and sensitized to apoptosis. Mutation of Asn-112, Asn-258, Asn-290, or Asn-311 to glutamine modestly reduced apoptosis resistance, while mutation of Asn-322 or simultaneous mutation of the other four asparagine residues caused a loss of cytoprotection by FGFR4. None of the glycomutants altered the migration of cancer cells. Finally, mutation of Asn-112 caused a partial localization of FGFR4 to the Golgi. Overall, preventing glycosylation at individual residues reduced the cell survival function of FGFR4 and receptor glycosylation may regulate access to an extracellular protease or proteolytic susceptibility of FGFR4.

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

The BRAF kinase is a critical node in the MAPK signaling pathway and is mutated in approximately 8% of human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), occurring in half of malignant melanomas. This mutation hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. BRAF inhibitors including vemurafenib, dabrafenib and encorafenib have produced impressive responses in V600X patients, however resistance usually emerges within a year, including RAS mutation, BRAFV600E amplification, and BRAFV600E intragenic deletion or splice variants. These inhibitors are also ineffective against non-V600 BRAF mutants (Class II & III). To address some of these limitations we have developed CFT1946, a bifunctional degradation activating compound (BiDAC™) degrader comprising a BRAF kinase domain targeting ligand linked to a cereblon ligand. CFT1946 is capable of degrading BRAF V600E (Class I), G469A (Class II), G466V (Class III) mutations, and the p61-BRAFV600E splice variant while maintaining exquisite selectivity against the proteome including WT BRAF and CRAF. In A375 cells, CFT1946 potently degraded BRAFV600E (Emax = 26%; DC50 = 14nM at 24hr) and, inhibited ERK phosphorylation (IC50 = 11nM at 24hr) and cell growth (GI50 = 94nM at 96hr) while having no effect in the mutant KRAS driven cell line HCT116. In A375 xenografts, oral delivery of CFT1946 resulted in deeper tumor regressions when dosed at 10 mg/kg PO BID and compared favorably to a clinically relevant dose of encorafenib. We further evaluated CFT1946 in an engineered A375-BRAFV600E/NRASQ61K double mutant model of BRAF inhibitor resistance. CFT1946 was able to degrade BRAFV600E in these cells and was much more effective than encorafenib at inhibiting viability in vitro. In this model, in vivo dosing of single agent CFT1946 caused robust tumor growth inhibition and combination with the MEK inhibitor, trametinib, resulted in tumor regressions. The combination of encorafenib and trametinib showed no activity in the same model. Next, we demonstrated that CFT1946 was able to degrade additional BRAF mutant proteins including G469A (Class II), G466V (Class III), and the p61-BRAFV600E splice variant using heterologous expression in HEK293T cells. Additionally, we also showed that CFT1946, but not encorafenib, inhibited proliferation of the BRAFG466V heterozygous lung tumor cell line H1666. Based on its activity in preclinical models, including models of BRAF inhibitor resistance, and its drug-like properties we are progressing CFT1946 as a candidate for clinical development in patients with solid tumors bearing BRAF V600X mutations. Further, given CFT1946’s activity on non-V600 BRAF mutations, we are continuing to explore CFT1946 and related BiDAC degraders as therapeutic options for patients bearing Class II or Class III BRAF mutations. Citation Format: Mathew E. Sowa, Bridget Kreger, Joelle Baddour, Yanke Liang, Jeffrey R. Simard, Laura Poling, Ping Li, Robert Yu, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe Sahil Patel, Richard Deibler, Kyle S. Cole, Scott Eron, David Cocozziello, Fazlur Rahman, Moses Moustakim, Christopher G. Nasveschuk, Katrina L. Jackson, Mark Fitzgerald, Victoria Garza, Morgan O’Shea, Gesine Veits, Jeremy L. Yap, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Stewart L. Fisher, Roy M. Pollock. Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2158.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Background Cutaneous squamous cell carcinoma (SCC) is the second most common type of skin cancer. Only 2% to 5% of SCCs metastasize; however, those do carry a poor prognosis. Immunohistochemistry (IHC) is widely used by pathologists to characterize skin cancers and provide clinically useful information. Objective To evaluate the potential prognostic associations between IHC findings and metastasis in SCC. Methods Searches were conducted in MEDLINE via PubMed for articles published between 1999 and 2019. Search criteria included key words "immunohistochemistry" and "cutaneous squamous cell carcinoma." Six hundred and fifty-three articles were returned and screened, which ultimately left 31 for inclusion in our manuscript. Results Thirty-one articles analyzed in this review included a discussion of the expression of a particular IHC marker and the associated risk of metastasis and/or clinical utility of IHC markers in SCC, especially metastatic SCC. Markers that had several or more studies supporting clinical utility were E-cadherin, podoplanin, CD8+ T cells, PD-L1, epidermal growth factor receptor, and Cyclin D1. Conclusion Immunohistochemistry profiling of SCC may be useful in select cases when providing a prognosis remains challenging and in identification of potential therapeutic targets for high-risk or metastatic tumors.

The performance of a wavelength-division multiplexing (WDM) free-space optical (FSO) communication system in a turbulent atmosphere employing optical amplifiers to improve capacity is investigated, in the presence of amplified spontaneous emission noise, scintillation, beam spreading, atmospheric attenuation and interchannel crosstalk. Using on-off keying modulation, Monte Carlo simulation techniques are used to obtain the average bit error rate and system capability due to scintillation and the effect of introducing a power control algorithm (PCA) to the system is investigated. The PCA ensures that at any receiving instant, the same turbulence-free powers are received by all the receiving lenses. The performance of various WDM FSO communication system configurations such as non-amplified systems with an adaptive decision threshold (NOAADT), nonamplified systems with a non-adaptive decision threshold, fixed gain amplified systems with an adaptive decision threshold, fixed gain amplified systems with a nonadaptive decision threshold and saturated gain amplified systems with a non-adaptive decision threshold (SOANADT) are investigated. Results obtained show that the SOANADT is superior to the NOAADT and the PCA is only beneficial in amplified systems.

A synthesis of two structurally related macrodiolides representing the aglycone of natural products elaiophylin and halichoblelide is described. The key transformation for both is a Ti(II)-mediated (silyloxy)enyne cyclization, generating a new methyl stereocenter and providing a diene that can be selectively cross metathesized with crotonic acid.

Massive deployment of free space optical (FSO) communication systems in future optical access networks will use wavelength division multiplexing (WDM) technology to leverage extended reach and enhanced user capacity. An analytical framework for the estimation of outage probability in a turbulence-affected WDM FSO system is presented in this study. The authors derive simplified expressions of the probability of outage in the presence of turbulence-accentuated interchannel crosstalk, and show how design parameters such as the FSO link length, demultiplexer adjacent channel rejection and receiver aperture diameter affect the performance of the system. Numerical results show that the outage probability due to irradiance fluctuation is worsened in WDM FSO systems by the effects of turbulence-affected interchannel crosstalk signals. Specifically, the average crosstalk power and the degree of irradiance fluctuations on the crosstalk set a limit on the outage probability performance of the system which appears in the form of outage floors.

The interplay between platelets and tumor cells is known to play important roles in metastasis by enhancing tumor cell survival, tumor-vascular interactions, and escape from immune surveillance. However, platelet-covered circulating tumor cells (CTC) are extremely difficult to isolate due to masking or downregulation of surface epitopes. Here we describe a microfluidic platform that takes advantage of the satellite platelets on the surface of these "stealth" CTCs as a ubiquitous surface marker for isolation. Compared to conventional CTC enrichment techniques which rely on known surface markers expressed by tumor cells, platelet-targeted isolation is generally applicable to CTCs of both epithelial and mesenchymal phenotypes. Our approach first depletes unbound, free platelets by means of hydrodynamic size-based sorting, followed by immunoaffinity-based capture of platelet-covered CTCs using a herringbone micromixing device. This method enabled the reliable isolation of CTCs from 66% of lung and 60% of breast cancer (both epithelial) patient samples, as well as in 83% of melanoma (mesenchymal) samples. Interestingly, we observed special populations of CTCs that were extensively covered by platelets, as well as CTC-leukocyte clusters. Because these cloaked CTCs often escape conventional positive and negative isolation mechanisms, further characterization of these cells may uncover important yet overlooked biological information in blood-borne metastasis and cancer immunology.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1–5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

The performance of a free-space optical (FSO) communication system in a turbulent atmosphere employing an optical amplifier (OA) cascade to extend reach is investigated. Analysis of both single and cascaded OA FSO communication links is given and the implications of using both adaptive (to channel state) and non-adaptive decision threshold schemes are analysed. The benefits of amplifier saturation, for example in the form of effective scintillation reduction when a non-adaptive decision threshold scheme is utilised at the receiver for different atmospheric turbulence regimes, are presented. Monte Carlo simulation techniques are used to model the probability distributions of the optical signal power, noise and the average bit error rate due to scintillation for the cascade. The performance of an adaptive decision threshold is superior to a non-adaptive decision threshold for both saturated and fixed gain preamplified receivers and the ability of a saturated gain OA to suppress scintillation is only meaningful for system performance when a non-adaptive decision threshold is used at the receiver. An OA cascade can be successfully used to extend reach in FSO communication systems and specific system implementations are presented. The optimal cascade scheme with a non-adaptive receiver would use frequent low gain saturated amplification.

A hybrid fibre and free-space optical communication link using digital pulse-position modulation (DPPM) in a wavelength-division-multiplexing system is proposed. Such a system, which could provide a power efficient, robust and flexible solution to high-speed access networks, is a contender for a passive optical network solution and could readily be deployed in areas with restrictions in optical fibre installation, or alternatively as a disaster recovery network. Interchannel crosstalk and atmospheric turbulence are major impairments in such a system and could combine in some cases to degrade the system. Both impairments are investigated here and the results are presented in the form of bit error probability, required optical transmission power and power penalties. Depending on the position of the interferer relative to the desired user, power penalties of about 0.2–3.0 dB for weak turbulence and above 20 dB for strong turbulence regimes are reported for bit error rate of 10−6. DPPM scheme with a coding level of 2 show about 2 dB improvements over on–off-keying scheme.

A chemokine's sugary release As immune cells survey the body for pathogens, they circulate through the blood and migrate through the lymphatic system. The latter route allows for tissues and lymph nodes—the central hubs of the immune system—to communicate. Kiermaier et al. reveal the importance of the monosaccharide sialic acid in keeping immune cells in motion. Multiple sialic acids decorate the surface CCR7 on immune cells. CCR7 recognizes proteins called chemokines, which direct where cells move in the body. Sialic acids on CCR7 release one such chemokine present on lymph node endothelial cells from an inhibited state, allowing immune cells to enter lymph nodes. Science , this issue p. 186

Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB–mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9–TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.

Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the α-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and β3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.

Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.

We study the mechanism of the population inversion in mid-infrared fibre lasers based on a chalcogenide glass host doped with active lanthanide ions. Three lanthanide dopant ions are considered: terbium, dysprosium and praseodymium. We predict the relevant trivalent ion level populations and gain. The simulation parameters were obtained by fabricating and optically characterising a series of trivalent ion doped chalcogenide glass samples. We also provide simple analytical expressions that aid the design of the cascade lasing process.

Wavelength division multiplexing (WDM) has been proposed for fibre, intersatellite, free space and indoor optical communication systems. Digital pulse position modulation (DPPM) is a more power efficient modulation format than on-off keying (OOK) and a strong contender for the modulation of free-space systems. Although DPPM obtains this advantage in exchange for a bandwidth expansion, WDM systems using it are still potentially attractive, particularly for moderate coding levels. However, WDM systems are susceptible to interchannel crosstalk and modelling this in a WDM DPPM system is necessary. Models of varying complexity, based on simplifying assumptions, are presented and evaluated for the case of a single crosstalk wavelength. For a single crosstalk, results can be straightforwardly obtained by artificially imposing the computationally convenient constraint that frames (and thus slots also) align. Multiple crosstalk effects are additionally investigated, for the most practically relevant cases of modest coding level, and using both simulation and analytical methods. In general, DPPM maintains its sensitivity advantage over OOK even in the presence of crosstalk while predicting lower power penalty at low coding level in WDM systems.

Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF-16a, a putative ortholog of Caenorhabditis elegans DAF-16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF-16 is involved in the insulin/IGF-I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggests that B. malayi DAF-16a is a member of the FOXO family of forkhead proteins.

Determining the best way to efficiently use limited water resources, for food and energy-dedicated crops, has become crucial due to the rise in extreme events (floods/droughts) and higher variability in rainfall attributed to global climate change. Changing climate conditions will require new crops to be adapted to a changing agricultural environment. Reliable information on seasonal trends in crop growth and evapotranspiration with associated uncertainty/confidence ranges is crucial to guide the development of new crops and management strategies to cope with future climate. Given that crop growth is strongly coupled to soil moisture, developing reliable growth curves requires a detailed understanding of soil moisture at the field-scale. Typically, it is impractical to collect soil samples to adequately assess soil moisture that represents both spatial distribution at the field-scale and temporal dynamics on the scale of a growing season (e.g. 110days for cereals). A novel way to address soil moisture monitoring challenges is through an integrated, agro-ecosystems-level approach using an integrated sensing system that can link data from multiple platforms (wireless sensors, satellites, airborne imagery, near real-time climate stations). Assimilated data can, then, be fed into predictive models to generate reference crop growth curves and predict regionally-specific yield potentials. However, integrated sensing requires interagency cooperation, common data processing standards and long-term, timely access to data. Large databases need to be reusable by various organizations and accessible, in the future, with comprehensive metadata. During the 2012 growing season a feasibility study was conducted which involved measuring field-scale soil moisture with sensor network technology. The experiment utilized radially-distributed sensors for tracking in-season soil moisture. OpenGIS-compliant services and standards were utilized to provide long-term access to sensor data and construct corresponding metadata. Sensor Model Language, an inter-operable metadata format, was used to create documentation for the sensor system and sensing components. Two different third party implementations of the Sensor Observation Service were tested for providing long-term access to the data. This work discusses a set of key recommendations for monitoring field-scale soil moisture dynamics for integration with remote sensing and models, including: (1) Improved in situ sensing technology that would allow for less restrictive soil moisture measurements. (2) Integration of field-scale in situ networks with regional remote sensing monitoring. (3) The development of software and web services to integrate data from multiple sources with models for decision support.

A model that accounts for the switching operation of a photonic crystal micro-cavity is presented. The model accounts for the bipolar drift-diffusion in the micro-cavity. The impact of surface recombination on the surface charge and carrier transport is modelled self-consistently. The optical fields in the micro-cavity are modelled using coupled-mode theory.

A synthesis of carolacton, a myxobacterial natural product that has profound effects on Streptococcus mutans biofilms, is reported. The synthesis proceeds via a longest linear sequence of 14 steps from an Evans β-ketoimide and enabled preliminary evaluations of the effects of late-stage intermediates on S. mutans biofilms. These studies suggest that further investigations into carolacton's structure-function relationships are warranted.

Cholangiocarcinoma (CCA) is a terminal diagnosis, with 5-year survival rates averaging less than 10%. Thus, developing therapeutic options for cholangiocarcinoma patients is essential. FGFR4 is a heavily N-glycosylated receptor tyrosine kinase that signals through Akt, ERK and STAT3 pathways, and whose overexpression in cancer has previously been observed. The activation and signaling through FGFR4 involve the intracellular tyrosine kinase domain that is activated upon ligand binding the extracellular receptor region. Examination of expression in the TCGA database and our studies in CCA cells demonstrated a role for FGFR4 in promoting tumor cell survival and proliferation. Further, we have observed the presence of a proteolytic cleavage product of FGFR4, that is comprised of the intracellular kinase domain, which we call R4-ICD. Our data and previous studies on recombinant protein demonstrated that the intracellular kinase domain in the absence of the transmembrane and ligand-binding domains is constitutively active. HuCCT-1 cells (human CCA cell line lacking endogenous FGFR4 expression) stably transfected to express R4-ICD had increased proliferation and pro-survival phenotypes when compared to the parental cell line. Of 14 frozen CCA human tumor samples, all showed expression of R4-ICD. Compared to normal liver tissue, R4-ICD expression was greater in 65% (9 of 14) of tumor samples than in normal liver. In addition, total FGFR4 expression was lower in normal cholangiocytes than human CCA samples. Full length FGFR4 expression was observed in 12 tumor samples (85%). Of note, full-length FGFR4 expression levels were far weaker than R4-ICD expression in the 10 samples that showed both signals. Because R4-ICD is produced from full length receptor, we interpret the predominance and higher prevalence of R4-ICD to reflect efficient processing to a stable intracellular kinase fragment. We demonstrated at least two glycoforms of FGFR4, terminal and core, in cell lines, and these were also observed in human tumors. We hypothesized that glycosylation may regulate R4-ICD proteolytic production. Live CCA cells in culture were treated with PNGase F to remove extracellular N-linked sugars from cell surface proteins, including FGFR4. Deglycosylation of FGFR4 was confirmed by immunoblotting. Upon deglycosylation, R4-ICD levels rapidly increased. In summary, CCA cells in culture and in tumors were demonstrated to have high levels of R4-ICD, a constitutively active kinase that promoted tumor cell survival and proliferation. The stable cleavage product of FGFR4, R4-ICD, was increased on receptor deglycosylation. Whether aberrant FGFR4 glycosylation contributes to R4-ICD predominance in tumor samples or to tumor aggressiveness remains to be tested. Further understanding of R4-ICD formation will allow us to better target CCA, possibly by reducing proteolytic processing in conjunction with kinase inhibition. Citation Format: Andrew J. Phillips, Ashley M. Mohr, Mary Anne Phillippi, Justin L. Mott, Keith R. Johnson. N terminal glycosylation regulates FGFR4 cleavage in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 856.

The human Mediator complex is a central integrator for transcription and represents a primary interface that allows DNA-binding transcription factors to communicate their regulatory signals to the RNA polymerase II enzyme. Because Mediator is dynamic both in terms of subunit composition and structure, it presents challenges as a target for small molecule probes. Moreover, little high-resolution structural information exists for Mediator. Its global requirement for transcription, as well as its distinct, transcription factor specific interaction surfaces, however, suggest that development of probes that bind specific Mediator subunits might enable gene- and pathway-specific modulation of transcription. Here we provide a brief overview of the Mediator complex, highlighting biological and structural features that make it an attractive target for molecular probes. We then outline several chemical strategies that might be effective for targeting the complex.

A short total synthesis of routiennocin methyl ester is reported. The key reaction is the direct acylation of a titanacyclopropane with valerolactone to give a ketone.

The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrum small tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation. QC 20130524

Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5 (YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50 for SIK2 inhibition for > 16 hours and reduce phosphorylation of a known SIK substrate in vivo. While in vivo active doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2 knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivo, and further support the development of SIK inhibitors for treatment of inflammatory disorders.

Soil freeze-thaw cycles play an important role in all aspects of agro-ecosystems, such as crop productivity, the evolution of the soil matrix, including trace-gas emissions. In regions that experience synoptic weather conditions throughout the winter, freeze-thaw cycles generally occur in one of two categories; seasonal or winter cycles. Current soil vegetation atmosphere models (SVAT’s) often include a heat-transport soil freeze-thaw algorithm, but lack detail on complex interactions between the main driving variables. Boundary conditions for these models are often based only on a few climate variables and typically lack regional context. A nested statistical analysis was applied to identify the optimal set of environmental variables (via a stepwise regression selection procedure) to track soil freeze-thaw dynamics. Historical data collected between the years 2006-2009, for 17 long-term climate stations distributed across southern Alberta Canada was utilized. Cross-correlation between wind speed and maximum air temperature identified Chinook-driven freeze-thaw events, with such interaction varying significantly across the region and by soil depth. Climate-soil interactions were most significant predictors of soil temperature during winter months. The seasonal freeze-thaw cycle is estimated to vary between 112 - 131 days, consisting of 12 - 20 winter cycles (1 cm depth), and 1-5 winter cycles (5 cm depth) with average lag time of 26 - 112 days. Freeze-thaw prediction was greatly improved when higher-order climate interaction terms were considered. Our findings highlight the importance for soil-water and more complex ecosystem, SVAT models to better resolve regional-driven climatic trends. Alongside improved representation of regional trends aimed at reducing model-based uncertainty, such efforts are expected to, in tandem, help advance the geostatistical design, and implementation of agroenvironmental monitoring systems that combine in-situ and satellite/remote-sensing derived estimates of near-surface soil moisture.

The fish pathogenic oomycete Saprolegnia parasitica causes the disease Saprolegniosis in salmonids and other freshwater fish, resulting in considerable economic losses in aquaculture. Very little is known about the molecular and cellular mechanisms underlying the infection process of fish pathogenic oomycetes. In order to investigate the interaction in detail, an in vitro infection assay using an Oncorhynchus mykiss (rainbow trout) cell line (RTG-2) was developed. In a zoospore/cyst cDNA library, we identified the ORF SpHtp1, which encodes a secreted protein containing an RxLR motif. Detailed expression analysis indicated that SpHtp1 is highly expressed in zoospores/cysts from S. parasitica and in the very early stages of infection on RTG-2 cells, when compared with in vitro-grown mycelium. Moreover, the protein, SpHtp1, was found to translocate into the RTG-2 trout cells, during the interaction with S. parasitica, and also when the RTG-2 cells were treated with recombinant SpHtp1 fused to a C-terminal His-tag. These findings suggest that protein translocation could play an important role in Saprolegniosis.

Performance evaluations, for bit error rate (BER) and power penalty, are performed for DC coupled burst mode (BM) receivers in the presence of both amplified spontaneous emission (ASE) noise and interchannel crosstalk. Such BM receivers acquire the threshold from preamble bits, so the threshold is a random variable, because of contamination by noise and crosstalk. Here such a crosstalk contaminated threshold is described using a moment generating function for the first time. This facilitates the first use of Chernoff bound, modified Chernoff bound and saddle-point approximation techniques for BM reception under these conditions. A Gaussian approximation (GA) is also calculated. Results for an optically preamplified receiver indicate that the overall penalty (threshold acquisition penalty in the presence of crosstalk (and ASE)) exceeds the straightforward summation of the separate crosstalk and threshold acquisition penalties (in dB) and while this difference is particularly pronounced for the GA it remains significant for the other techniques. Furthermore, the GA is seen to be more pessimistic in BER compared to the other approaches and less accurate (as it exceeds an upper bound). Relative to the other techniques, the penalty predicted by the GA is optimistic for low crosstalk but pessimistic for high crosstalk.

The bit-error-rate (BER) performance of an optically preamplified peak detecting burst mode (BM) receiver is analyzed. The analysis takes into account amplified spontaneous emission (ASE) noise and, for the first time with this receiver architecture, interchannel crosstalk. Furthermore, the ASE beat noises are more appropriately included through the use of a noncentral chi-square (NCCS) distribution in contrast to earlier attempts, which used a Gaussian approximation (GA), though a GA is also developed here for the interchannel crosstalk case, for comparative purposes. Results are presented in terms of BER and penalty curves. The peak detector-based BM receiver shows larger threshold acquisition (TA) penalty than an integrator-based BM receiver in the presence of ASE. Also ASE beat noise modeling using the NCCS distribution shows greater power penalty for interchannel crosstalk than does the GA. The sum of separate crosstalk and TA penalties is found to be less than the combined TA penalty obtained in the presence of interchannel crosstalk.

Experimental investigation of the role of interband effects in four-wave mixing (FWM) in semiconductor optical amplifiers (SOAs) is reported. Carrier density pulsations (CDP) in SOAs, caused by optical wave beating, are measured in the RF domain. This gives strong experimental confirmation of the link between CDP and wave mixing, as usually assumed in theory of FWM in SOAs. The dependence of CDP amplitude on bias current and optical power is also established.

A DC coupled burst-mode receiver for digital pulse position modulation (DPPM) is proposed for the first time. An analysis of the potential performance benefits of using such a receiver configuration in the upstream of a passive optical network is given. An optical pre-amplifier is assumed. Bit error rate expressions are derived and the performance is evaluated in terms of sensitivity and threshold acquisition penalty. Results are compared with a DC-coupled on–off keyed non-return-to-zero burst-mode receiver, and it is seen that DPPM's continuous mode sensitivity advantage (about 8 dB for the optimal case) is only reduced to 7.7 dB when overhead preamble is restricted to 12 bits and 6 dB when restricted to 6 bits. Thus, if necessary, DPPM can almost recover its sensitivity advantage by a trade off with preamble length. As such DPPM can potentially offer an increase in optical network unit numbers by a factor of 4 or, alternatively, an increase in range of around 20 km.

A novel method for optical signal-to-noise ratio monitoring is presented. The method is based on two-filter narrowband filtering which, combined with the knowledge of the modulation format, allows the determination of the amount of amplified spontaneous emission noise in the channel spectrum. The major advantage of the method is simplified experimental setup. The robustness of the method against various signal impairments is discussed and assessed through simulation. It is shown that the method provides sufficient accuracy for most practical applications and is remarkably resilient against other physical phenomena reducing signal quality. In particular, the method is inherently insensitive to chromatic dispersion and polarisation-related impairments. All results are presented for 10 Gb/s return-to-zero format.

Bit error rate and threshold acquisition penalty evaluation is performed for an optically preamplified DC-coupled burst mode receiver using a moment generating function (MGF) description of the signal plus noise. The threshold itself is a random variable and is also described using an appropriate MGF. Chernoff bound (CB), modified Chernoff bound (MCB) and the saddle-point approximation (SPA) techniques make use of the MGF to provide the performance analyses. This represents the first time that these widely used approaches to receiver performance evaluation have been applied to an optically preamplified burst mode receiver and it is shown that they give threshold acquisition penalty results in good agreement with a prior existing approach, whilst having the facility to incorporate arbitrary receiver filtering, receiver thermal noise and non-ideal extinction ratio. A traditional Gaussian approximation (GA) is also calculated and comparison shows that it is clearly less accurate (it exceeds the upper bounds provided by CB and MCB) in the realistic cases examined. It is deduced, in common with the equivalent continuous mode analysis, that the MCB is the most sensible approach. Copyright © 2009 John Wiley & Sons, Ltd.