In Vitro Model of Prepacked Carbon Dioxide Absorber Use: Development and Testing.

BACKGROUND: Carbon dioxide absorbers allow the use of fresh gas flow below minute ventilation (V˙E). Models are developed and tested in vitro to quantify their performance with variable carbon dioxide load (V˙CO2), fresh gas flow, V˙E, end-tidal carbon dioxide (ETco2) fraction, and the type of workstation used. METHODS: First principles are used to derive a linear relationship between fresh gas flow and fractional canister usage or FCU0.5 (the reciprocal of the time for the inspiratory carbon dioxide fraction to reach 0.5%). This forms the basis for two basic models in which V˙E was measured by spirometry or calculated. These models were extended by multiplying V˙E with an empirical workstation factor. To validate the four models, two hypotheses were tested. To test whether the FCU0.5 intercept varied proportionally with V˙CO2 and was independent of V˙E, FCU was measured for 10 canisters tested with a fixed 0.3 l/min fresh gas flow and a range of V˙CO2 while V˙E was either constant or adjusted to maintain ETco2 fraction. A t test was used to compare the two groups. To confirm whether a change in V˙CO2 accompanied by a change in V˙E to maintain ETco2 fraction would shift the linear fresh gas flow-FCU0.5 relationship in a parallel manner, 19 canisters were tested with different combinations of V˙CO2 and fresh gas flow. These measured FCU values were compared to those predicted by the four models using Varvel's performance criteria. RESULTS: With 0.3 l/min fresh gas flow, FCU0.5 was proportional with V˙CO2 and independent of whether V˙E was adjusted to maintain ETco2 fraction or not (P = 0.962). The hypothesized parallel shift of the fresh gas flow-FCU0.5 relationship was confirmed. Both extended models are good candidate models. CONCLUSIONS: The models predict prepacked canister performance in vitro over the range of V˙E, fresh gas flow, and V˙CO2 likely to be encountered in routine clinical practice. In vivo validation is still needed.

Malt1 self-cleavage is critical for regulatory T cell homeostasis and anti-tumor immunity in mice.

Mucosa-associated lymphoid tissue 1 (Malt1) regulates immune cell function by mediating the activation of nuclear factor κB (NF-κB) signaling through both its adaptor and proteolytic function. Malt1 is also a target of its own protease activity and this self-cleavage further contributes to NF-κB activity. Until now, the functional distinction between Malt1 self-cleavage and its general protease function in regulating NF-κB signaling and immune activation remained unclear. Here we demonstrate, using a new mouse model, the importance of Malt1 self-cleavage in regulating expression of NF-κB target genes and subsequent T cell activation. Significantly, we further establish that Treg homeostasis is critically linked to Malt1 function via a Treg intrinsic and extrinsic mechanism. TCR-mediated Malt1 proteolytic activity and self-cleavage was found to drive Il2 expression in conventional CD4+ T cells, thereby regulating Il2 availability for Treg homeostasis. Remarkably, the loss of Malt1-mediated self-cleavage alone was sufficient to cause a significant Treg deficit resulting in increased anti-tumor immune reactivity without associated autoimmunity complications. These results establish for the first time that inhibition of MALT1 proteolytic activity could be a viable therapeutic strategy to augment anti-tumor immunity.

The XPO1 Inhibitor KPT-8602 Synergizes with Dexamethasone in Acute Lymphoblastic Leukemia.

PURPOSE: KPT-8602 (Eltanexor) is a second-generation exportin-1 (XPO1) inhibitor with potent activity against acute lymphoblastic leukemia (ALL) in preclinical models and with minimal effects on normal cells. In this study, we evaluated whether KPT-8602 would synergize with dexamethasone, vincristine, or doxorubicin, three drugs currently used for the treatment of ALL. EXPERIMENTAL DESIGN: First, we searched for the most synergistic combination of KPT-8602 with dexamethasone, vincristine, or doxorubicin in vitro in both B-ALL and T-ALL cell lines using proliferation and apoptosis as a readout. Next, we validated this synergistic effect by treatment of clinically relevant B- and T-ALL patient-derived xenograft models in vivo. Finally, we performed RNA-sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the mechanism of synergy. RESULTS: KPT-8602 showed strong synergism with dexamethasone on human B-ALL and T-ALL cell lines as well as in vivo in three patient-derived ALL xenografts. Compared with single-drug treatment, the drug combination caused increased apoptosis and led to histone depletion. Mechanistically, integration of ChIP-seq and RNA-seq data revealed that addition of KPT-8602 to dexamethasone enhanced the activity of the glucocorticoid receptor (NR3C1) and led to increased inhibition of E2F-mediated transcription. We observed strong inhibition of E2F target genes related to cell cycle, DNA replication, and transcriptional regulation. CONCLUSIONS: Our preclinical study demonstrates that KPT-8602 enhances the effects of dexamethasone to inhibit B-ALL and T-ALL cells via NR3C1- and E2F-mediated transcriptional complexes, allowing to achieve increased dexamethasone effects for patients.

Safe targeting of T cell acute lymphoblastic leukemia by pathology-specific NOTCH inhibition.

Given the high frequency of activating NOTCH1 mutations in T cell acute lymphoblastic leukemia (T-ALL), inhibition of the γ-secretase complex remains an attractive target to prevent ligand-independent release of the cytoplasmic tail and oncogenic NOTCH1 signaling. However, four different γ-secretase complexes exist, and available inhibitors block all complexes equally. As a result, these cause severe "on-target" gastrointestinal tract, skin, and thymus toxicity, limiting their therapeutic application. Here, we demonstrate that genetic deletion or pharmacologic inhibition of the presenilin-1 (PSEN1) subclass of γ-secretase complexes is highly effective in decreasing leukemia while avoiding dose-limiting toxicities. Clinically, T-ALL samples were found to selectively express only PSEN1-containing γ-secretase complexes. The conditional knockout of Psen1 in developing T cells attenuated the development of a mutant NOTCH1-driven leukemia in mice in vivo but did not abrogate normal T cell development. Treatment of T-ALL cell lines with the selective PSEN1 inhibitor MRK-560 effectively decreased mutant NOTCH1 processing and led to cell cycle arrest. These observations were extended to T-ALL patient-derived xenografts in vivo, demonstrating that MRK-560 treatment decreases leukemia burden and increased overall survival without any associated gut toxicity. Therefore, PSEN1-selective compounds provide a potential therapeutic strategy for safe and effective targeting of T-ALL and possibly also for other diseases in which NOTCH signaling plays a role.

HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616-31. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.