Transient inhibition of the JAK/STAT pathway prevents B-ALL development in genetically predisposed mice

Preventing development of childhood B-cell acute lymphoblastic leukemia (B-ALL), a disease with devastating effects, is a longstanding and unsolved challenge. Heterozygous germline alterations in the PAX5 gene can lead to B-ALL upon accumulation of secondary mutations affecting the JAK/STAT signaling pathway. Preclinical studies have shown that this malignant transformation occurs only under immune stress such as exposure to infectious pathogens. Here we show in Pax5+/− mice that transient, early-life administration of clinically relevant doses of ruxolitinib, a JAK1/2 inhibitor, significantly mitigates the risk of B-ALL following exposure to infection; 1 of 29 animals treated with ruxolitinib developed B-ALL versus 8 of 34 untreated mice. Ruxolitinib treatment preferentially targeted Pax5+/− versus wild-type B-cell progenitors and exerted unique effects on the Pax5+/− B-cell progenitor transcriptional program. These findings provide the first in vivo evidence for a potential strategy to prevent B-ALL development.C. Cobaleda and C. Vicente-Dueñas labs are members of the EU COST Action LEGEND (CA16223). Research in C. Vicente-Dueñas group has been funded by Instituto de Salud Carlos III through the project " PI17/00167 and by a “Miguel Servet Grant” [CPII19/00024 - AES 2017-2020; co-funded by European Regional Development Fund (ERDF)/European Social Fund (ESF) "A way to make Europe"/"Investing in your future"]. J.J. Yang and K.E. Nichols receive funding from the American Lebanese Syrian Associated Charities (ALSAC) and R01CA241452 from the NCI. Research in ISG group is partially supported by FEDER and by SAF2015-64420-R MINECO/FEDER, UE, RTI2018-093314-B-I00 MCIU/AEI/FEDER, UE, 9659122185-122185-4-21 MCIU/AEI/FEDER, UE, by Junta de Castilla y León (UIC-017, CSI001U16, CSI234P18, and CSI144P20). M. Ramírez-Orellana and I. Sánchez-García have been supported by the Fundacion Unoentrecienmil (CUNINA project). C. Cobaleda, M. Ramírez-Orellana, and I. Sánchez-García have been supported by the Fundación Científica de la Asociación Española contra el Cáncer (PRYCO211305SANC). A. Casado-García (CSI067-18) and M. Isidro-Hernández (CSI021-19) are supported by FSE-Conserjería de Educación de la Junta de Castilla y León 2019 and 2020 (ESF, European Social Fund) fellowship, respectively. J. Raboso-Gallego is supported by a scholarship from University of Salamanca co-financed by Banco Santander and ESF. S. Alemán-Arteaga is supported by an Ayuda para Contratos predoctorales para la formación de doctores (PRE2019-088887)

A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues.

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo-like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions

Continuous low-dose infusion of patupilone increases the therapeutic index in mouse and rat tumour models

Patupilone is a microtubule-targeted cytotoxic agent with clinical efficacy, but causes diarrhoea in more than 80% of patients. The efficacy and tolerability of patupilone delivered continuously by subcutaneous (s.c.) mini-pumps [(mini-pump dose (MPD)] or by intravenous bolus administration [intravenous bolus dose (IVBD)] were compared preclinically to determine whether the therapeutic index could be improved. The antiproliferative potency in vitro of patupilone was determined by measuring total cell protein. Tumours were grown s.c. in rats (A15) or nude mice (KB31, KB8511) or intracranially in nude mice (NCI-H460-Luc). Efficacy was monitored by measuring tumour volumes, bioluminescence or survival. Toxicity was monitored by body weight and/or diarrhoea. Total drug levels in blood, plasma, tissues or dialysates were quantified ex-vivo by liquid chromatography-mass spectroscopy/mass spectroscopy. Patupilone was potent in vitro with GI50s of 0.24-0.28 nmol/l and GI90s of 0.46-1.64 nmol/l. In rats, a single IVBD of patupilone dose dependently inhibited the growth of A15 tumours, but also caused dose-dependent body weight loss and diarrhoea, whereas MPD achieved similar efficacy, but no toxicity. In mice, MPD showed efficacy similar to that of IVBD against KB31 and KB8511 tumours, but with reduced toxicity. In a mouse intracranial tumour model, IVBD was more efficacious than MPD, consistent with patupilone concentrations in the brain. MPD provided constant plasma levels, whereas IVBD had very high C 0/C min ratios of 70-280 (rat) or 8000 (mouse) over the dosing cycle. Overall, the correlation of plasma and tumour levels with response indicated that a Cave of at least GI90 led to tumour stasis. Continuous low concentrations of patupilone by MPD increased the therapeutic index in s.c. rodent tumour models compared with IVBD by maintaining efficacy, but reducing toxicity

A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo–like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.ISSN:1663-981

JAK1/2 and pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease”

JAK inhibitors have demonstrated rapid and durable reductions in splenomegaly, as well as improvement in symptoms and quality of life in patients with myelofibrosis. However, the impact on the mutant allele burden and bone marrow fibrosis has been modest, indicating that combinations with other agents may further improve outcomes. Histone deacetylase inhibition has emerged as a promising combination modality based on in vitro studies using JAK2V617F mutant models that suggested a synergistic effect upon combination with a JAK2 inhibitor, and encouraging single-agent activity of the pan-deacetylase inhibitor panobinostat in phase I/II myelofibrosis trials. Here, we investigated the combination of the JAK1/2 inhibitor ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. The combination was found to have a more profound effect on efficacy readouts as compared to either agent alone, and the analysis of pharmacodynamic readouts demonstrated that ruxolitinib and panobinostat have non-overlapping and complementary effects on biological pathways

JAK1/2 and pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease

JAK inhibitors have demonstrated rapid and durable reductions in splenomegaly, as well as improvement in symptoms and quality of life in patients with myelofibrosis. However, the impact on the mutant allele burden and bone marrow fibrosis has been modest, indicating that combinations with other agents may further improve outcomes. Histone deacetylase inhibition has emerged as a promising combination modality based on in vitro studies using JAK2V617F mutant models that suggested a synergistic effect upon combination with a JAK2 inhibitor, and encouraging single-agent activity of the pan-deacetylase inhibitor panobinostat in phase I/II myelofibrosis trials. Here, we investigated the combination of the JAK1/2 inhibitor ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. The combination was found to have a more profound effect on efficacy readouts as compared to either agent alone, and the analysis of pharmacodynamic readouts demonstrated that ruxolitinib and panobinostat have non-overlapping and complementary effects on biological pathways

JAK1/2 and pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease

JAK inhibitors have demonstrated rapid and durable reductions in splenomegaly, as well as improvement in symptoms and quality of life in patients with myelofibrosis. However, the impact on the mutant allele burden and bone marrow fibrosis has been modest, indicating that combinations with other agents may further improve outcomes. Histone deacetylase inhibition has emerged as a promising combination modality based on in vitro studies using JAK2V617F mutant models that suggested a synergistic effect upon combination with a JAK2 inhibitor, and encouraging single-agent activity of the pan-deacetylase inhibitor panobinostat in phase I/II myelofibrosis trials. Here, we investigated the combination of the JAK1/2 inhibitor ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. The combination was found to have a more profound effect on efficacy readouts as compared to either agent alone, and the analysis of pharmacodynamic readouts demonstrated that ruxolitinib and panobinostat have non-overlapping and complementary effects on biological pathways

K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition <em>In Vivo</em>

<div><p>Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.</p> </div