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Supplementary Data from TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis

Members of the Bcl-2 family play a major role in the pathobiology of head and neck cancer. We have shown that Bcl-2 orchestrates a cross talk between tumor cells and endothelial cells that have a direct effect on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly up-regulated in the tumor-associated endothelial cells compared with the endothelial cells of normal oral mucosa in patients with HNSCC. Here, we evaluated the effect of TW-37, a small-molecule inhibitor of Bcl-2, on the cell cycle and survival of endothelial cells and HNSCC and on the progression of xenografted tumors. TW-37 has an IC50 of 1.1 μmol/L for primary human endothelial cells and averaged 0.3 μmol/L for head and neck cancer cells (OSCC3, UM-SCC-1, and UM-SCC-74A). The combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, compared with single drug treatment. Notably, whereas cisplatin led to an expected G2-M cell cycle arrest, TW-37 mediated an S-phase cell cycle arrest in endothelial cells and in HNSCC. In vivo, TW-37 inhibited tumor angiogenesis and induced tumor apoptosis without significant systemic toxicities. Combination of TW-37 and cisplatin enhanced the time to tumor failure (i.e., 4-fold increase in tumor volume), compared with either drug given separately. Collectively, these data reveal that therapeutic inhibition of Bcl-2 function with TW-37 is sufficient to arrest endothelial cells and HNSCC in the S phase of the cell cycle and to inhibit head and neck tumor angiogenesis.[Mol Cancer Ther 2009;8(4):893–903]

Supplementary Figures from TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis

Increases in ambient salinity demand parallel increases in intestinal base secretion for maintenance of osmoregulatory status, which is likely the cause of a transient acidosis following transfer of euryhaline fish from freshwater to seawater. It was predicted that transfer of the marine Gulf toadfish (Opsanus beta) from seawater (35 ppt) to hypersaline (60 ppt) seawater (HSW) would lead to a transient acidosis that would be compensated by increases in branchial acid excretion to offset the acid–base disturbance. Toadfish exposed to HSW showed a significant decrease in blood pH and [HCO3−] but no increase in pCO2, followed by a full recovery after 48–96 h. A similar metabolic acidosis and recovery was found when fish were exposed to 60-ppt HCO3−-free seawater (HEPES-buffered), which may suggest that compensation for intestinal base loss during hypersaline treatment is from gill H+ excretion rather than gill HCO3− uptake. However, we cannot rule out that reduced branchial HCO3− excretion contributed to an increase in net acid excretion. Since colchicine prevents full compensation, translocation of H+ and/or HCO3− transporters between cytosolic compartments and plasma membrane fractions might be involved in compensating for the hypersalinity-induced acidosis. Translocation of transporters rather than de novo synthesis may represent a faster and less energetically demanding response to rapidly fluctuating and high salinities encountered by toadfish in their natural environment.

Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavourable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant, euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs – the gills and intestine. Using immunofluorescence, we localized aquaporin-3 protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial aquaporin-3 mRNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter aquaporin-3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish aquaporin-3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater, and facilitates intestinal permeability in seawater and freshwater.

Figures and Tables

[Graphic][1] Music is a common language across many cultures and is often celebrated with large festivals that can attract thousands of people in just a single day. For festival-goers, the relentless melodies and rhythms can be exhilarating, but for nearby neighbours, it can be an intrusive

[Graphic][1] In the Canadian North Pole, there exists but one freshwater fish species: the Arctic char ( Salvelinus alpinus ). This keystone species is an indicator of ecosystem health, because it is highly sensitive to environmental changes. And, because many northerners depend on char for

Increases in ambient salinity demand parallel increases in intestinal base secretion for maintenance of osmoregulatory status, which is likely the cause of a transient acidosis following transfer of euryhaline fish from freshwater to seawater. It was predicted that transfer of the marine Gulf toadfish (Opsanus beta) from seawater (35 ppt) to hypersaline (60 ppt) seawater (HSW) would lead to a transient acidosis that would be compensated by increases in branchial acid excretion to offset the acid-base disturbance. Toadfish exposed to HSW showed a significant decrease in blood pH and [HCO

[Graphic][1] Plastic pollution is one of the most pervasive and sinister ecological threats worldwide. Most of us are aware of animals ingesting and dying from plastic waste. However, much less is known of the more insidious problem caused by much smaller microplastics, which are formed

[Graphic][1] Some years ago, after putting out the recycling, I had the unpleasant discovery that my dog had got into my bag of jalapeno potato chips and thrown up all over the floor. They obviously hadn't sat well with him. As I cleaned up the mess, I pondered why my dog was so quick to

[Graphic][1] Within the Seychelles archipelago of the Indian Ocean is found the world's second-largest pristine coral atoll: Aldabra. Because of its geographical isolation and the difficulties accessing it, Aldabra has long been protected from human interference. Given this unique

[Graphic][1] If you're the kind of person who enjoys watching the perfectly choreographed singing of the von Trapp children in The Sound of Music, then the sight of translucent stink bugs crawling forth simultaneously from their eggs might be the stuff of nightmares. Other people, such as

Teleosts living in seawater continually absorb water across the intestine to compensate for branchial water loss to the environment. The present study reveals that the Gulf toadfish ( Opsanus beta) rectum plays a comparable role to the posterior intestine in ion and water absorption. However, the posterior intestine appears to rely more on SLC26a6 (a HCO3−/Cl−antiporter) and the rectum appears to rely on NKCC2 (SLC12a1) for the purposes of solute-coupled water absorption. The present study also demonstrates that the rectum responds to renoguanylin (RGN), a member of the guanylin family of peptides that alters the normal osmoregulatory processes of the distal intestine, by inhibited water absorption. RGN decreases rectal water absorption more greatly than in the posterior intestine and leads to net Na+and Cl−secretion, and a reversal of the absorptive short-circuit current ( ISC). It is hypothesized that maintaining a larger fluid volume within the distal segments of intestinal tract facilitates the removal of CaCO3precipitates and other solids from the intestine. Indeed, the expression of the components of the Cl−-secretory response, apical CFTR, and basolateral NKCC1 (SLC12a2), are upregulated in the rectum of the Gulf toadfish after 96 h in 60 ppt, an exposure that increases CaCO3precipitate formation relative to 35 ppt. Moreover, the downstream intracellular effects of RGN appear to directly inhibit ion absorption by NKCC2 and anion exchange by SLC26a6. Overall, the present findings elucidate key electrophysiological differences between the posterior intestine and rectum of Gulf toadfish and the potent regulatory role renoguanylin plays in osmoregulation.

This file contains supplementary results and tables of test statistics (Tables S2 to S7)

Freshwater- and seawater-acclimated Fundulus heteroclitus were exposed to acute hypoxia (10% air saturation, 3 h), followed by normoxic recovery (3 h). In both salinities, ventilation increased and heart rate fell in the classic manner, while Ṁ(O(2)) initially declined by ∼50%, with partial restoration by 3 h of hypoxia, and no O(2) debt repayment during recovery. Gill paracellular permeability (measured with [(14)C] PEG-4000) was 1.4-fold higher in seawater, and declined by 50% during hypoxia with post-exposure overshoot to 188%. A similar pattern with smaller changes occurred in freshwater. Drinking rate (also measured with [(14)C] PEG-4000) was 8-fold higher in seawater fish, but declined by ∼90% during hypoxia in both groups, with post-exposure overshoots to ∼270%. Gill diffusive water flux (measured with (3)H(2)O) was 1.9-fold higher in freshwater fish, and exhibited a ∼35% decrease during hypoxia, which persisted throughout recovery, but was unchanged during hypoxia in seawater fish. Nevertheless, freshwater killifish gained mass while seawater fish lost mass during hypoxia, and these changes were not corrected during normoxic recovery. We conclude that this hypoxia-tolerant teleost beneficially reduces gill water permeability in a salinity-dependent fashion during hypoxia, despite attempting to simultaneously improve Ṁ(O(2)), but nevertheless incurs a net water balance penalty in both freshwater and seawater.

[Graphic][1] Cruising at top speeds of 93 km h−1 (move over, Usain Bolt!), there aren't many fish that swim faster than mahi-mahi. Like high-performing athletes, mahi-mahi have exceptionally conditioned hearts that allow them to swim so swiftly. Yet, these extraordinary ocean athletes

The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (

The physiological effects of guanylin (GN) and uroguanylin (UGN) on fluid and electrolyte transport in the teleost fish intestine have yet to be thoroughly investigated. In the present study, the effects of GN, UGN, and renoguanylin (RGN; a GN and UGN homolog) on short-circuit current ( Isc) and the transport of Cl−, Na+, bicarbonate (HCO3−), and fluid in the Gulf toadfish ( Opsanus beta) intestine were determined using Ussing chambers, pH-stat titration, and intestinal sac experiments. GN, UGN, and RGN reversed the Iscof the posterior intestine (absorptive-to-secretory), but not of the anterior intestine. RGN decreased baseline HCO3−secretion, but increased Cl−and fluid secretion in the posterior intestine. The secretory response of the posterior intestine coincides with the presence of basolateral NKCC1 and apical cystic fibrosis transmembrane conductance regulator (CFTR), the latter of which is lacking in the anterior intestine and is not permeable to HCO3−in the posterior intestine. However, the response to RGN by the posterior intestine is counterintuitive given the known role of the marine teleost intestine as a salt- and water-absorbing organ. These data demonstrate that marine teleosts possess a tissue-specific secretory response, apparently associated with seawater adaptation, the exact role of which remains to be determined.

Background: The role of Minimal residual disease (MRD) as surrogate for survival in Multiple Myeloma (MM) patients is well established. Therefore, new response criteria recommend including Multiparameter Flow cytometry (MFC) and next generation Sequencing (NGS) MRD negativity (minimum sensitivity of 1 in 105 nucleated cell) to deeply characterize complete remission (CR) [Kumar SK, Lancet Oncol 2016]. Here we analyzed and compared MRD data from the FORTE trial both by MFC and NGS techniques. Methods: Newly diagnosed MM patients ≤65 years were randomized to receive carfilzomib, lenalidomide, dexamethasone (KRd) induction - autologous stem cell transplantation (ASCT) intensification - KRd consolidation (KRd_ASCT); KRd12 and carfilzomib, cyclophosphamide, dexamethasone (KCd) induction - ASCT intensification - KCd consolidation (KCd_ASCT). Thereafter, patients were randomized to maintenance therapy with lenalidomide alone or lenalidomide plus carfilzomib. MRD evaluation was performed by 8-color second generation flow cytometry (sensitivity 10-5) in patients who achieved at least a very good partial response (VGPR) before maintenance. In a subgroup of these ≥ VGPR patients, next generation flow (NGF; sensitivity 10-5 - 10-6) was performed. Moreover, in patients achieving ≥ CR, MRD pre-maintenance was also assessed by NGS (Adaptive Biotechnologies, Seattle, WA; sensitivity 10-5 - 10-6). Therefore, only patients achieving ≥ CR have both MFC and NGS evaluations, and we focused on this ≥ CR population to compare MRD results with the 2 techniques. Spearman's rank correlation coefficient was used to measure the concordance between MFC and NGS. Fisher or Pearson's Chi-squared tests were adopted, where appropriate, to evaluate the statistical significance, at the level of 0.05. Results: ≥ CR pre-maintenance was achieved in 233 patients enrolled in the trial; at data cut-off, MFC and NGS data were available for 176/233 (76%) patients who were included in this analysis (62 received KRd_ASCT, 65 KRd12 and 49 KCd_ASCT). Median age of this ≥ CR population is 57 years (IQR 52-62), 14% have International Staging System (ISS) III stage and 26% high risk cytogenetics by FISH features [either del(17p) or t(4;14) or t(14;16)] reflecting baseline clinical features of the entire FORTE population. 139/176 (79%) ≥ CR patients were MFC negative at a cut-off of at least 10-5. Rate of MRD negative MFC among ≥ CR negative in the 3 arms were: 53/62 (85%) in KRd_ASCT, 51/65 (78%) in KRd12 and 35/49 (71%) in KCd_ASCT, reflecting the higher rate of MCF MRD negativity pre-maintenance reported by ITT in the overall population in the 2 KRD arms [Gay F, ASH 2018]. NGS negativity at a cut-off of at least 10-5 was detected in 87/176 (49%) of ≥ CR patients. Rate of MRD negative NGS at least 10-5 among ≥ CR negative in the 3 arms were: 35/62 (56%) in KRd_ASCT, 31/65 (48%) in KRd12 and 21/49 (43%) in KCd_ASCT. NGS negativity at a cut-off 10-6 was detected in 34/123 (28%) of ≥ CR patients (for 53/176 CR patients 10-6 sensitivity was not reached). Rate of MRD negative NGS at least 10-6 among ≥ CR negative in the 3 arms were:14/41 (34%) in KRd_ASCT, 10/44 (23%) in KRd12 and 10/38 (26%) in KCd_ASCT. Thereafter, we have analyzed the concordance between MRD results by the two techniques. Overall (samples at 10-5 and 10-6 sensitivity), Spearman's coefficient is 0.63 (p < 0.001); discordances between the two methods have been observed in 54/176 paired samples (30%). In particular, 53 samples (30%) are NGS positive but MFC negative, of which 34/53 (64%) have reached 10-6 sensitivity by NGS. Only 1 MFC positive sample is NGS negative. In a subgroup of patients evaluable both by NGS and NGF (26 paired samples, sensitivity 10-6), Spearman' s coefficient is 0.83 (p < 0.001), although a higher sample size is needed to confirm these preliminary concordant results. Conclusion: In patients who achieved ≥ CR, rate of at least 10-5 MRD MCF negativity was 79% and rate of at least 10-5 NGS negativity was 49%. Assessment both by MFC and NGS showed a good concordance, particularly if the same sensitivity is reached. Longer follow up is needed to assess the impact of MFC in comparison with NGS on patients' outcomes, particularly to evaluate if 10-6 NGS or NGF sensitivity may provide further clinical information, possibly identifying patients with very long survival or potentially cured. Disclosures Oliva: Celgene: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Belotti:Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Galli:Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Leadiant (Sigma-Tau): Honoraria; Janssen: Honoraria; Celgene: Honoraria. Offidani:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vozella:Amgen: Honoraria; Celgene: Honoraria. Zambello:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kirsch:Adaptive Biotechnologies: Employment. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Ballanti:Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Corradini:Roche: Honoraria; Sanofi: Honoraria; KiowaKirin: Honoraria; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; BMS: Other: Travel Costs; Servier: Honoraria; Takeda: Honoraria, Other: Travel Costs; Gilead: Honoraria, Other: Travel Costs; AbbVie: Consultancy, Honoraria, Other: Travel Costs; Amgen: Honoraria; Celgene: Honoraria, Other: Travel Costs; Daiichi Sankyo: Honoraria; Jazz Pharmaceutics: Honoraria; Janssen: Honoraria, Other: Travel Costs. Omedé:Janssen: Membership on an entity's Board of Directors or advisory committees. Cavo:takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Musto:Amgen: Honoraria; Celgene: Honoraria. Boccadoro:Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding. Gay:AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The presentation includes discussion of off-label use of a drug or drugs for the treatment of multiple myeloma.

Introduction: The autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel (Axi-cel) improved long-term survival of patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the pivotal ZUMA-1 trial indicates a 2-year PFS of ~40% (Locke, Lancet Oncology 2018). Early identification of patients with increased relapse risk may allow for early intervention and improved outcomes. In a pilot study of 6 ZUMA-1 patients, minimal residual disease (MRD) evaluation via a next-generation sequencing MRD assay (Adaptive Biotechnologies, Seattle, WA) to assess for circulating tumor (ct)DNA, mirrored clinical outcome as assessed by PET-CT (Hossain et. al. Leukemia & Lymphoma 2019). Based on these promising results, a multi-institutional prospective study utilizing cell-free MRD assessments to predict outcomes in r/r DLBCL patients after Axi-cel therapy was initiated. Methods: To identify tumor clonotype(s), tumor DNA extracted from archival paraffin-embedded tissue underwent PCR amplification of IgH-VDJ, IgH-DJ and IgKappa/Lambda regions using universal consensus primers. CtDNA levels were measured pre-LD, 0, 7, 14, 21, 28, 56, 90, 180, 270, and 365 days following Axi-cel infusion. PET-CT scans were obtained at baseline, Day 28, Month 3, 6, and 12 with response assessed per Lugano criteria. Deauville 1-3 was considered PET-negative. The protocol prespecified that patients with less than Day 28 follow-up be excluded from analysis. Any detectable ctDNA was considered MRD positive. Results: Here we report on the pre-planned analysis of the first 50 study patients with at least a Day 28 MRD assessment and 3 months of follow up. An additional 4 patients with at least 3 months of follow-up but who did not have a Day 28 MRD assessment were also included. Baseline characteristics and clinical outcomes of patients were similar to ZUMA-1 and a real-world analysis of 295 patient who received Axi-cel (Nastoupil et al ASH 2018). The median age was 61 years old (range 19-76) (53.7% male, 46.3% female) and 59% of patients received 3 or more prior lines of therapy (range 1-6). After a median follow-up of 7.5 months, the best overall response rate was 87% (47 of 54) and complete response rate was 57% (31 of 54). The median OS was not reached and median PFS was 4.6 months (panel A). At Day 28, 56% (28 of 50) of patients were MRD negative (MRD-neg) and 44% (22 of 50) were MRD positive (MRD-pos). As compared to MRD-pos, MRD-neg correlated with improved median PFS (not reached vs. 2.96 months, p Conclusion: MRD monitoring using high-throughput sequencing of ctDNA has the potentially to make an impact on the clinical management of patients undergoing Axi-cel therapy. Furthermore, ctDNA is an informative tool to compare CAR19 therapies that vary by costimulatory domains or production methods. This technology potentially overcomes fundamental limitations of DLBCL imaging (cost, radiation exposure & limited repetition) and may minimize the need for surveillance PET-CT scans. These results provide a rationale for designing MRD-based risk-adaptive CAR T cell clinical trials. Figure Disclosures Kirsch: Adaptive Biotechnologies: Employment. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Mullins:Adaptive Biotechnologies: Employment. Lee:Adaptive Biotechnologies: Employment, Equity Ownership. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Locke:Cellular BioMedicine Group Inc.: Consultancy; Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor. Miklos:Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding.

For some species of ectothermic vertebrates, early exposure to hypoxia during embryonic development improves hypoxia-tolerance later in life. However, the cellular mechanisms underlying this phenomenon are largely unknown. Given that hypoxic survival is critically dependent on the maintenance of cardiac function, we tested the hypothesis that developmental hypoxia alters cardiomyocyte physiology in a manner that protects the heart from hypoxic stress. To test this hypothesis, we studied the common snapping turtle, which routinely experiences chronic developmental hypoxia and exploits hypoxic environments in adulthood. We isolated cardiomyocytes from juvenile turtles that embryonically developed in either normoxia (21% O2) or hypoxia (10% O2), and subjected them to simulated anoxia and reoxygenation, while simultaneously measuring intracellular Ca2+, pH and reactive oxygen species (ROS) production. Our results suggest developmental hypoxia improves cardiomyocyte anoxia-tolerance of juvenile turtles, which is supported by enhanced myofilament Ca2+-sensitivity and a superior ability to suppress ROS production. Maintenance of low ROS levels during anoxia might limit oxidative damage and a greater sensitivity to Ca2+could provide a mechanism to maintain contractile force. Our study suggests developmental hypoxia has long-lasting effects on turtle cardiomyocyte function, which might prime their physiology for exploiting hypoxic environments.

Philadelphia-like (Ph-like) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a subgroup of BCP-ALL with an expression pattern similar to BCR-ABL+ BCP-ALL that is associated with poor prognosis. Aberrant expression of CRLF2 in BCP-ALL constitutes the majority of Ph-like BCP-ALL cases. CRLF2 is a receptor subunit that together with the IL7RA subunit comprises the receptor of the proinflammatory cytokine TSLP. Though activation of the IL7R pathway is commonly associated with T-cell malignancies, we previously described IL7RA-activating mutations in BCP-ALL predominantly in the context of CRLF2 aberrant expression (Shochat C. et al. J. Exp. Med. 2011). Here we aimed to test the role of aberrations in CRLF2 and IL7RA in the development of Ph-Like BCP-ALL. Both CRLF2 and TSLP differ extensively between mice and human in amino acid sequence and in lineage expression pattern; loss-of-function germline mutations in IL7RA are associated with lack of B and T cells in mice but with lack of only T cells in humans. Hence, we chose to test the hypothesis that activation of CRLF2/IL7RA contributes to the development of Ph-like BCP-ALL in the context of human lymphopoiesis by using a human xenograft system. To aberrantly activate TSLP/IL7 signaling, we transduced cord-blood (CB) CD34+ hematopoietic progenitors with a set of lentiviral vectors carrying CRLF2 and/or IL7RA [(wild type (IL7RAwt) or IL7R bearing an activating mutation (IL7RAins)] under a B-cell promoter/enhancer (to accentuate B-cell lineage expression). The backbone vector (BB) expressing GFP was used as a control. Transduced CB cells were transplanted into NOD/LtSz-scid IL2Rγnull (NSG) mice and engrafted cells were analyzed 24-30 weeks after transplantation. To test for self-renewal capacity, BM cells from primary engrafted mice were serially transplanted into secondary recipients and the occurrence of human engraftment was tested 24-30 weeks after transplantation. Enforced expression of activated IL7RA with or without CRLF2 led to a significant block in B-cell development at the B-cell progenitor stage (CD19+CD10+sIgM-) in vivo resembling the differentiation stage of leukemic cells (figure Bi). Repertoire sequencing of CD10+CD19+-transduced cells that were sorted from BM of transplanted mice revealed a significantly higher population of DJ-rearranged cells in the CRLF2-IL7RAins-transduced population than in BB-transduced cells (mean ratio of DJ/total rearrangement: BB:0.35+/-0.024, CRLF2-IL7RAins:0.76+/- 0.07, p=0.039, n=3 paired cord blood), in agreement with the early differentiation block phenotype measured by immunophenotyping. These cells furthermore exhibited a Ph-like gene expression pattern when compared to BB-transduced cells in gene set enrichment analysis. Overexpression of IL7RA alone significantly enhanced the early-B fraction (CD19+CD10+CD34+) in the BM of transplanted mice (figure Bii). Additionally, aberrant expression of IL7RA enhanced self-renewal capacity as was evident by an increased ability of the transduced cells to engraft in secondary recipients (number of mice with detectable human engraftment out of secondary transplanted mice: BB:0/6, CRLF2-IL7RAwt:0/3, CRLF2-IL7RAinst:0/3, IL7RAwt-GFP:3/6, IL7RAins-GFP:5/8). Notably, in one case, secondary transplantation of IL7RAins-transduced CB triggered the development of acute BCP-ALL. The leukemic cells (CD19+CD10+CD34+sIgM-) were clonal as validated by V(D)J rearrangement (figure Cii), had the ability to further propagate in serial transplantations and gained secondary Ph-like BCP-ALL-characteristic chromosomal deletions in the short arm of chromosome 9 (in the region including the genes for CDKN2A/B, PAX5 and JAK2) and the short arm of chromosome 7 (the region including IKZF1) (figure Ciii). These results support the hypothesis that aberrant activation of the CRLF2/IL7RA pathway in human B-cell lineage progenitors creates a pre-leukemic state by arresting differentiation of B-cell progenitors, instating Ph-like expression pattern and inducing self-renewal. This is the first model of de novo Ph-like BCP-ALL development from normal human hematopoietic progenitors in vivo. Additionally, we present here a first direct in vivo demonstration of a role for IL7 in human B-cell development. Disclosures No relevant conflicts of interest to declare.

The Reaction of phenylmercury(II) acetate with salicylaldehyde morpholine N-thiohydrazone (H2smth) and 2-hydroxyacetophenone morpholine N-thiohydrazone (H2apmth) in dry ethanol under gentle refluxing condition form light yellow complexes [PhHg(Hsmth)] (1) and [PhHg(Hapmth)] (2) involving formation of Hg-S bond from the thiol form of the ligands after deprotonation of the SH proton. The structures of the complexes as determined by X-ray crystallography shows that the complex (1) has a distorted T-shaped geometry while the complex (2) adopts familiar linear coordination geometry. Complex (2) has two independent molecules comprising the asymmetric unit. Both the complexes form two-dimensional supramolecular assemblies due to a combination of weak intermolecular Hg····π and Hg····Hg interactions. The Hg····π and Hg····Hg distances are 3.937 and 4.0216(10) A, respectively, possibly indicating weak mercuriophilic interactions. The luminescent properties of the complexes in solution and in the solid state at room temperature are also described.

Background Early response to induction chemotherapy is a significant prognostic factor in the outcome of children with acute lymphoblastic leukemia (ALL). High throughput sequencing (HTS) of rearranged immune receptor (TCR and Ig) genes offers the possibility of a more accurate, sensitive, and standardized approach to determination of early response to therapy.In this study, we investigate the ability of an HTS assay to risk stratify children with B-ALL at the end of induction therapy in comparison with flow cytometry (FC), assess the impact of increased MRD sensitivity on risk group assignment, evaluate the significance of MRD discordance between HTS and FC, and identify a novel subset of patients having an inferior outcome. Methods 619 paired Pretreatment and End of Induction (Day 29) samples from patients with B-ALL enrolled on Children's Oncology Group (COG) clinical trials AALL0331 (standard-risk, SR) and AALL0232 (high-risk, HR) having minimal residual disease (MRD) at Day 29 of less than 0.1% by flow cytometry were assayed by high throughput sequencing of CDR3 regions of IGH and TCRG. Dominant clonal CDR3 sequences in the pretreatment samples were quantitated in the paired Day 29 samples as residual disease of total nucleated cells without knowledge of the FC results. The relationship of residual disease determined by HTS and FC to 5-year event-free and overall survival (EFS and OS) was evaluated using Kaplan-Meier statistics. Results HTS detected a dominant clonal sequence in 93.2% of Pretreatment B-ALL samples, providing an informative cohort of standard-risk (N=282) and high-risk (N=297) patients. Using a threshold of 0.01% on the combined cohort, HTS and FC show identical EFS and OS for MRD positive (77.7% ± 0.04, 91.6% ± 0.03) and negative (92.5% ± 0.02, 96.3% ± 0.01) subsets, see Figure 1. Interestingly, reducing the HTS threshold from 0.01% to 0.0001% results in an improvement in EFS for the HTS MRD positive subset in both standard (80.1% -> 88.2%) and high-risk (75.3% -> 84.8%) patients, likely due to major reductions in the number of patients otherwise scored as MRD negative using the higher threshold of 0.01%(70.9% -> 27.0% SR and 78.5% -> 36.7% HR). This reflects the much more favorable outcome of the large cohort of patients with MRD between 0.0001% and 0.01% compared to those >0.01%. Little improvement in EFS is seen for HTS MRD negative patients with a reduction in MRD threshold. Maximal difference in EFS is achieved at an HTS threshold of 0.01%. Importantly, the subset of SR patients with no detectable residual clonal sequence at any level (19.9% of total) show an excellent EFS (98.1% ± 0.02) and OS (100% ± 0), different from the similar subset of HR patients (30.0% of total) showing less favorable EFS (92.7% ± 0.04) and OS (95.1% ± 0.03). Patients discordant for MRD at a threshold of 0.01%, either HTS+/FC- (N=55) or HTS-/FC+ (N=17), show intermediate EFS compared with concordantly positive or negative patients. Of interest, patients lacking a detectable clonal IgH sequence (N=42) show a significantly inferior EFS (78.5% ± 0.08 vs. 89.3% ± 0.02, p=0.01) but not OS. Conclusions HTS is equivalent to FC in its ability to risk stratify patients with childhood B-ALL at End of Induction therapy using a MRD threshold of 0.01%. Reducing the HTS MRD threshold below 0.01% does not improve risk stratification, but does allow identification of a small subset of MRD negative standard-risk patients virtually certain to be cured with current therapy. Patients discordant for MRD between HTS and FC have an outcome intermediate between that seen for concordant patients. Patients lacking a detectable clonal IgH sequence, presumably representing a more primitive form of leukemia, show a significantly inferior outcome. Figure 1. Equivalence of outcomes by high throughput sequencing and flow cytometry for B-ALL patients at a residual disease threshold of 0.01%. Figure 1. Equivalence of outcomes by high throughput sequencing and flow cytometry for B-ALL patients at a residual disease threshold of 0.01%. Disclosures Wood: Pfizer: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement. Kirsch:Adaptive Biotechnology: Employment. Crossley:Adaptive: Employment, Equity Ownership. Williamson:Adaptive Biotechnology: Employment. Borowitz:HTG Molecular: Consultancy; BD Biosciences: Research Funding; Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Robins:Adaptive Biotechnology: Employment.

Members of the Bcl-2 family play a major role in the pathobiology of head and neck cancer. We have shown that Bcl-2 orchestrates a cross talk between tumor cells and endothelial cells that have a direct effect on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly up-regulated in the tumor-associated endothelial cells compared with the endothelial cells of normal oral mucosa in patients with HNSCC. Here, we evaluated the effect of TW-37, a small-molecule inhibitor of Bcl-2, on the cell cycle and survival of endothelial cells and HNSCC and on the progression of xenografted tumors. TW-37 has an IC50 of 1.1 μmol/L for primary human endothelial cells and averaged 0.3 μmol/L for head and neck cancer cells (OSCC3, UM-SCC-1, and UM-SCC-74A). The combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, compared with single drug treatment. Notably, whereas cisplatin led to an expected G2-M cell cycle arrest, TW-37 mediated an S-phase cell cycle arrest in endothelial cells and in HNSCC. In vivo, TW-37 inhibited tumor angiogenesis and induced tumor apoptosis without significant systemic toxicities. Combination of TW-37 and cisplatin enhanced the time to tumor failure (i.e., 4-fold increase in tumor volume), compared with either drug given separately. Collectively, these data reveal that therapeutic inhibition of Bcl-2 function with TW-37 is sufficient to arrest endothelial cells and HNSCC in the S phase of the cell cycle and to inhibit head and neck tumor angiogenesis.[Mol Cancer Ther 2009;8(4):893–903]

The aim of this study was to evaluate the long-term pharmacokinetics, safety, and efficacy of sertraline in children and adolescents with obsessive-compulsive disorder (OCD) or major depressive disorder (MDD).After 42-day initial treatment and 9-day withdrawal phases, children (6-12 years, n = 16) and adolescents (13-18 years, n = 27) entered a 24-week open-label phase, with sertraline titrated to 200 mg/day. Blood samples for plasma sertraline and N-desmethylsertraline levels were taken at the beginning of the 24-week phase and at weeks 1, 4, 8, 12, and 24. Efficacy and safety data were also collected.Mean maximum daily dose at endpoint was 157 +/- 49 mg. For female and male children, mean sertraline/N-desmethylsertraline concentrations normalized to a 200-mg dose were 85.0/160 ng/mL (n = 8) and 79.3/134 ng/mL (n = 8), respectively, and for female and male adolescents, 70.5/109 ng/mL (n = 16) and 76.3/120 ng/mL (n = 8). No significant age or gender effects or age-by-gender interactions were observed in sertraline values. Mean sertraline plasma concentrations normalized for dose and body weight did not differ significantly by age or gender. Three (3) patients (7%) discontinued owing to adverse events. In patients with OCD (n = 10), improvements were observed in Children's Yale-Brown Obsessive Compulsive Scale (CY-BOCS) (p = 0.029) and National Institute of Mental Health (NIMH) Global Obsessive Compulsive Scale (OCS) (p = 0.01). In MDD patients (n = 32), Clinical Global Impression (CGI) Severity (p = 0.002) and Improvement (p = 0.011) improved.Long-term treatment of MDD and OCD with sertraline up to 200 mg/day in children and adolescents results in dose-normalized plasma concentrations comparable to those seen in adults. Sertraline was generally well tolerated, and patients demonstrated clinical improvement over 24 weeks of treatment.

Complex engineering studies typically involve hundreds of analysis routines and thousands of variables. The sequence of operations used to evaluate a design strongly affects the speed of each analysis cycle. This influence is particularly important when numerical optimization is used, because convergence generally requires many iterations. Moreover, it is common for disciplinary teams to work simultaneously on different aspects of a complex design. This practice requires decomposition of the analysis into subtasks, and the efficiency of the design process critically depends on the quality of the decomposition achieved. This paper describes the development of software to plan multidisciplinary design studies. A genetic algorithm is used, both to arrange analysis subroutines for efficient execution, and to decompose the task into subproblems. The new planning tool is compared with an existing heuristic method. It produces superior results when the same merit function is used, and it can readily address a wider range of planning objectives.

It is shown that the creep buckling of slender or thin-walled structures whose material deforms elastically as well as in consequence of time-hardening creep can be analyzed first as if the deformations were due entirely to steady creep. The results of this analysis can be easily modified to account for time-hardening creep and this modification does not involve any approximation when the time-hardening creep strain rate is expressed as the product of a function of stress by a function of time. The effect of simultaneous linearly elastic deformations can be taken into account by multiplying the critical time obtained for steady creep by a numerical factor. This correction involves no farther-reaching approximations than the usual steady-creep buckling analysis itself.

This file contains supplementary materials and methods and a table of solutions used in the study (Table S1)

This file contains supplementary materials and methods and a table of solutions used in the study (Table S1)