Small Molecule Combinations Research Articles

CHIR99021 and Brdu Are Critical in Chicken iPSC Reprogramming via Small-Molecule Screening.

Background/Objectives: Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Methods: Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. Results: The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 104 cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. Conclusions: In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms.

Distinctive small molecules blend: Promotes lacrimal gland epithelial cell proliferation in vitro and accelerates lacrimal gland injury repair in vivo

PurposeThis study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for in vitro expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury. MethodsLGECs proliferative capacity was assessed by cell counting, crystal violet staining, qRT-PCR and immunofluorescence. Cell differentiation was achieved by manipulating culture conditions and assessed by qRT-PCR and AQP5 immunofluorescence. LGECs were seeded in Matrigel for three-dimensional culture and assessed by qRT-PCR and immunofluorescence. Secretory function of the cultures was assayed by ELISA. In vivo, 2C injection verified its reparative capacity in a mouse LG injury model. Corneal fluorescein staining, phenol red cotton thread, H&E, immunofluorescence and Western blot were used to assess LG injury repair. ResultsLGECs cultured with 2C exhibited high expression of stemness/proliferation markers and maintained morphology and proliferative capacity even after the tenth passage. Removal of 2C was efficacious in achieving LGECs differentiation, characterized by the increased AQP5 expression and LTF secretion. 3D spheroids cultured with 2C demonstrated differentiation potential, forming microglandular structures containing multiple LG cell types with secretory functions after 2C removal. In vivo, 2C improved the structural integrity and function of the injured LG. ConclusionsWe present a small molecule combination, 2C, that promotes LGECs expansion and differentiation in vitro and accelerates LG injury repair in vivo. This approach has potential applications for providing a stable source of seed cells for tissue engineering applications, providing new sights for LG-related diseases treatment.

Experimental study on small molecule combinations inducing reprogramming of rat fibroblasts into functional neurons.

To establish a methodological system for reprogramming rat embryonic fibroblasts (REF) into chemically induced neurons (ciNCs) via small molecule compounds to provide safe and effective donor cells for treatment of neurodegenerative diseases. Based on the method established by PEI Gang's research group to directly reprogram human fibroblasts into neurons, the induction medium and maturation medium was optimized by replacing the coating solution, mitigating oxidative stress injury, adding neurogenic protective factors, adjusting the concentration of trichothecenes, performing small-molecule removal experiments, and carrying out immunofluorescence and Western blotting on cells at different stages of induction to validate the effect of induction. When the original protocol was used for induction, the cell survival rate was (34.24±2.77)%. After replacing the coating solution gelatin with matrigel, the cell survival rate increased to (45.41±4.27)%; after adding melatonin, the cell survival rate increased to (67.95±5.61)% and (23.43±1.42)% were transformed into neural-like cells; after adding the small molecule P7C3-A20, the cell survival rate was further increased to (76.27±1.41)%, and (39.72±4.75)% of the cells were transformed into neural-like cells. When the concentration of trichothecene was increased to 30 μmol/L, the proportion of neural-like cells reached (55.79±1.90)%; after the removal of SP600125, (86.96±2.15)% of the cells survived, and the rate of neural-like cell production increased to (63.43±1.60)%. With the optimized protocol, REF could be successfully induced into ciNC through the neural precursor cell stage, in which the neural precursor cells were able to highly express the neural precursor cell markers SRY-related HMG-box gene 2 (Sox2) and paired box 6 (Pax6) as well as neuron-specific marker tubulin 1 (Tuj1), while the expression of fiber-associated protein vimentin was reduced. After two weeks of induction of neural precursor cells in a maturation medium, most cells displayed neuronal-like cell morphology. The induced ciNCs were able to highly express the mature neuronal surface markers Tuj1 and microtubule-associated protein 2 (MAP2), while the expression of vimentin was reduced. The small molecule combinations optimized in this study can reprogram REF to ciNCs under normoxic conditions.

WITHDRAWAL: Quantitative modeling of approved and emerging therapeutics for modifying TTR levels in patients with Transthyretin Amyloid Cardiomyopathy.

Withdrawal: McGirr, K., Sarkar, S., Subramanian, K. (2023) "Quantitative modeling of approved and emerging therapeutics for modifying TTR levels in patients with Transthyretin Amyloid Cardiomyopathy" CPT: Pharmacometrics & Systems Pharmacology. https://doi.org/10.1002/psp4.13078. The above article, published online on November 6, 2023, in Wiley Online Library (http://wileyonlinelibrary.com), has been withdrawn by agreement between the authors, journal Editor-in-Chief, France Mentré, the American Society for Clinical Pharmacology and Therapeutics (ASCPT), and Wiley Periodicals LLC. The withdrawal has been agreed to at the authors' request due to a disagreement over the ownership of some data contained therein.

The possible link between aluminum phosphide poisoning and NLRP3 inflammasome

Aluminum phosphide (AlP) is a fumigant used to control pests. However, human exposure to this substance can lead to poisoning. AlP reacts with gastric acid and water to release phosphine (PH3). This toxic gas affects the mitochondria and quickly enters the bloodstream. This gas impairs the mitochondria's ability to generate ATP, leading to oxidative stress, mitochondrial dysfunction, and damage to mitochondrial DNA (mt-DNA). Mitochondria release damage-associated molecular patterns (DAMPs) to shield cells from stress and stimulate the innate immune system.Nonetheless, if mitochondrial homeostasis is disrupted, an excessive immune response can occur, causing organ dysfunction. Mitochondrial reactive oxygen species (mt-ROS) and mt-DNA act as DAMPs. Upon recognition of DAMPs, the inflammasome is activated, causing inflammation. The nucleotide-binding domain, leucine-rich-containing family, and pyrin domain-containing-3 (NLRP3) gene on chromosome 1's extended arm encodes a protein that triggers the human inflammasome. NLRP3 is the most extensively researched inflammasome subfamily. It is essential to recognize that the pathophysiology of mitochondrial dysfunction and NLRP3 activation is complex and multifactorial, and a comprehensive approach may be needed to address both the underlying causes and downstream inflammatory responses effectively. The cGAS-STING pathway is essential for immune responses, and targeting specific nodes can be beneficial. Therapeutic approaches include small molecule drugs, biologics, vaccines, and combination therapies. Since there is a significant relationship between mitochondrial events and NLRP3 activation, targeting this inflammasome could be a promising strategy for developing antidotes for AlP poisoning.

Abstract 2048: Integrated tumor models for immune oncology: Using live cell imaging for prediction of treatment efficacy in vitro and in vivo

Abstract Background: The preclinical evaluation of novel cancer treatments demands comprehensive model systems in vitro that provide meaningful data before entering in vivo studies. Here we evaluate the capabilities of live cell imaging systems to evaluate novel immune therapies. Using integrated immune and tumor cell models in vitro we demonstrate, that these model systems can generate reliable data of pharmacodynamic activity of biologicals, small molecules or combinatorial approaches for further preclinical in vivo characterization. Methods: Target tumor cell killing was assessed in vitro with immune cells (T- and NK-cells) and engagers. Tumor cells were transduced with a fluorescent marker to discriminate tumor cells from immune cells. The technology was used to determine inhibition of cell motility (re-invasion) after scratching of tumor cell monolayers. Cells were monitored using the IncuCyte. Dose-response-curves of single treatments and all combinations were generated in parallel. Active therapies were selected for further in vivo validation of immune cell killing. Humanized mice were generated by injection of CD34+ HSC or human immune cell subsets. Immune cell engraftment was monitored by FACS. To analyze the effect biologicals or small molecules, tumor cells were transplanted into these humanized mice. Tumor development and therapeutic effects were monitored by BLI measurements. Results and conclusion: Tumor cell killing by immune cells and monolayer scratch assay in 96 well format were successfully monitored in the IncuCyte. Here, data can be generated over time without the need of new samples at every time point compared with conventional end point measurements. Using antibodies directing immune cells to attack target cells extensive cell killing was observed over time. These data predicted in vivo treatment outcome in mice co-engrafted with human immune cells. After successful humanization of mice, immune cells can be directed to kill target tumor cells. Small molecule combinations were tested in vitro utilizing the metastasis/2D scratch assay. After setup of dose-response curves for two molecules combinatorial treatments were tested. Here we found a synergistic increase in efficacy. These combinations were tested in vivo to evaluate their abilities to inhibit cell motility and distant metastasis. Here we show that the in vitro assays predicted correctly the highest efficacy of combined treatments compared to mono treatments.The IncuCyte System provides data that translate our integrated model systems into in vivo studies. We have shown that activated immune cells can kill target tumor cells in vitro. These data have been validated in vivo using immune cell humanized mice. Further, immune cells, biologicals and small molecule based treatments can be tested either alone or in combination, allowing the preselection of active combinations for further development. Citation Format: Dennis Kobelt, Maria Stecklum, Simone Rhein, Wolfgang Walther, Jens Hoffmann. Integrated tumor models for immune oncology: Using live cell imaging for prediction of treatment efficacy in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2048.

DIRECTEUR: transcriptome-based prediction of small molecules that replace transcription factors for direct cell conversion.

Direct reprogramming (DR) is a process that directly converts somatic cells to target cells. Although DR via small molecules is safer than using transcription factors (TFs) in terms of avoidance of tumorigenic risk, the determination of DR-inducing small molecules is challenging. Here we present a novel in silico method, DIRECTEUR, to predict small molecules that replace TFs for DR. We extracted DR-characteristic genes using transcriptome profiles of cells in which DR was induced by TFs, and performed a variant of simulated annealing to explore small molecule combinations with similar gene expression patterns with DR-inducing TFs. We applied DIRECTEUR to predicting combinations of small molecules that convert fibroblasts into neurons or cardiomyocytes, and were able to reproduce experimentally verified and functionally related molecules inducing the corresponding conversions. The proposed method is expected to be useful for practical applications in regenerative medicine. The code and data are available at the following link: https://github.com/HamanoLaboratory/DIRECTEUR.git.

Abstract LB_C03: Protein translation inhibition enforces histone deacetylase inhibitor activity resulting in synergistic pancreatic cancer cell death

Abstract Management of pancreatic cancer presents significant challenges due to late-stage diagnosis, aggressive tumor biology, and limited efficacy of existing treatment options. Gemcitabine and FOLFIRINOX (folinic acid, 5-FU, irinotecan hydrochloride, oxaliplatin) are standard treatments for advanced pancreatic cancer. Despite these therapies the overall 5-year survival rate for pancreatic cancer patients is at 12%, largely due to drug resistance. Thus, there is a critical need to explore new therapeutic strategies. Our study explores a small molecule combination that induces epigenetic reprogramming as a potential novel therapeutic strategy for pancreatic cancer. We have identified marked sensitivity of pancreatic cancer cells to the combination of a novel inhibitor of RNA helicase eIF4A, DMPatA (MZ735), and a histone deacetylase (HDAC) inhibitor, romidepsin. A short-term, low dose romidepsin treatment, when combined with MZ735, triggers persistent global histone acetylation, transcriptional upregulation, replication inhibition, and augmented DNA damage. This combined effect induces a synergistic anti-proliferative response in pancreatic cancer cells. We have identified a tolerable protocol for this combination regimen in animals, demonstrating its efficacy against tumor growth in a MIA PaCa-2 xenograft mouse model of pancreatic cancer. Treatment with the combination resulted in inhibition and then complete suppression of tumor growth in animals for 39 days. Our in vitro studies aimed at understanding the mechanism of this combination treatment showed that at low doses of romidepsin monotherapy, histone acetylation rapidly faded, while robust acetylation persisted to 18 and 24 hours with the combination. Additionally, R-loop accumulation was observed using confocal microscopy and DRIP-qPCR, along with a reduction in replication fork speed, which might account for the identified DNA damage and genome instability. The combination treatment significantly reduces acetyl-CoA and c-MYC protein levels and disrupts glycolysis and oxidative phosphorylation. We show that a structurally different HDAC inhibitor exhibits the same synergistic effects when co-administered with MZ735, confirming a unique method of eliciting synthetic lethality in PDAC cells. In conclusion, this combination shows significant antitumor activity in both in vitro and in vivo models of pancreatic cancer and warrant translation to the clinic. Citation Format: Maryam Safari, Luigi Scotto, Agnes Basseville, Thomas Litman, Haoran Xue, Luba Petrukhin, Ping Zhou, Christopher Damoci, Minhzhao Zhu, Kenneth Hull, Kenneth P Olive, Antonio T Fojo, Daniel Romo, Susan E Bates. Protein translation inhibition enforces histone deacetylase inhibitor activity resulting in synergistic pancreatic cancer cell death [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C03.

Methods of Estimating Renal Function for Use in Clinical Trial Eligibility Criteria Widely Vary in Phase II and III Clinical Trials in Acute Myeloid Leukemia

Background Renal function is an important parameter to inform drug-dosing in oncology. In acute myeloid leukemia (AML), commonly used therapies or their metabolites are renally cleared. Several equations are commonly used to estimate renal function, such as Cockcroft-Gault (CG) to estimate creatinine clearance (CrCl) and the Modification of Diet in Renal Disease (MDRD) or Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equations to calculate estimated glomerular filtration rates (eGFR). These equations frequently use endogenous serum markers, such as creatinine (Cr) and cystatin-C due to their ready availability but can have limitations. For example, Cr can be affected by muscle mass, diet, and age. Current ADDIKD international consensus guidelines recommend using the CKD-EPI creatinine formula to calculate eGFR to guide anti-cancer drug dosing unless direct GFR measurement is indicated. Renal function criteria are commonly used in clinical trial eligibility. In this study, we analyzed the types of reported renal eligibility criteria in phase II and III trials in AML and the methodology specified for renal function estimation, which can significantly impact treatment options for patients and potentially confound toxicity evaluation. Methods We examined the clinicaltrials.gov database to review publicly reported renal eligibility criteria for interventional phase II and III clinical trials recruiting adults with AML between 2021 to 2023. Trials including pediatric patients and exclusive phase I studies were excluded. Chi-square tests were used to evaluate for potential associations between eGFR cutoff criteria and disease variables. Results A total of 134 trials met criteria for review, including 84 (63%) trials specifically for AML and 50 (37%) allowing additional diagnoses. Of the total study cohort, 107 trials (80%) were evaluating chemotherapy, small molecule inhibitors, immunotherapy and/or combinations of these whereas 27 (20%) were evaluating primarily cell therapy and/or transplant-based regimens. Among the 134 trials, 21 (16%) did not mention renal or general organ function in eligibility criteria; 16 (12%) mentioned renal or organ function in inclusion/exclusion criteria but did not specify an eGFR cutoff value; and 15 (11%) specified a serum Cr cutoff value without a specific eGFR cutoff value. Among 82 (61%) studies specifying eGFR cutoffs: 18 required eGFR ≥60 ml/min and 64 specified cutoffs between 30 ml/min to 50 ml/min or greater (table 1). We found an association between studies requiring an eGFR of 60 ml/min or greater and studies investigating cell therapy or transplant-based regimens (p<0.01). We did not find associations between eGFR cutoff requirements and trials based on disease status (relapsed/refractory AML versus other AML states, p=0.24). We next evaluated methodologies reported in eligibility criteria to estimate renal function. Among the 113 trials commenting on renal function criteria: 15 (13%) did not report either an eGFR cutoff or methodology of determination; 67 (59%) specified eGFR cutoffs without specifying the methodology of determination and/or provided serum Cr-based cutoffs; and 31 (27%) specified either a method of direct measurement or equations to estimate eGFR (including CG-based in 25, CKD-EPI in 1, MDRD-based in 3 and radioisotope methodology in 1) (table 1). No trials mentioned cystatin-C as part of renal function determination. Conclusion In contemporary phase II/III clinical trials for adult patients with AML, we identified that while most trials include renal function in inclusion/exclusion criteria, there was tremendous heterogeneity in methodologies specified for determination of renal function which may make efficacy and toxicity comparisons challenging and impact patient eligibility across institutions. Several trials specified serum Cr cutoffs which can be impacted by factors besides renal function such as age or muscle mass. This study was limited by what trial criteria were publicly available and additional information specifying methodologies to calculate renal function may be available in full trial protocols. Further research is needed to determine the most suitable method for assessing eGFR in patients with AML to optimize treatment exposures for patients and to help reduce confounding of trial results or trial eligibility by institutional practice variation.

Drug Response Profiling Informs Personalized Bridging to Cell Therapy for Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia

Introduction Treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL) remains challenging in spite of recent progress with immunotherapy and targeted small-molecule combinations. Growing awareness of the molecular complexity of the disease with subtype-specific cancer dependencies positions functional drug testing as a promising precision tool to improve the prediction of clinical drug efficacy and adapt treatment. We leverage automated, image-based Drug Response Profiling (DRP) and machine learning to deliver individualized therapeutic options in real time for ALL patients with urgent medical needs. Here we report on the DRP experience of 307 primary samples from 278 patients with BCP-ALL, T-ALL/T-LBL and mixed phenotypic acute leukemia (MPAL) registered from 2016 to 2022 in a non-interventional setting. Methods We have generated a reference set of 141 diagnostic samples from patients with high-risk (75%) or standard/medium-risk (25%) disease as defined by the clinical trial AIEOP-BFM ALL 2017, alongside 122 samples with first and 44 with more advanced relapse. We assayed cell viability at single-cell level from viably frozen bone marrow (BM) samples, co-cultured with hTERT-immortalized mesenchymal stromal cells (MSC) and following a 72h drug incubation with a library of up to 110 compounds. We quantified cell counts at five concentrations in duplicates by segmenting and classifying MSC, live and dead leukemia cells using a modular image processing pipeline deployed on cloud infrastructure. We longitudinally confirmed assay consistency with PDX models and monitored integrity of the stromal layer on a per-sample basis. In a subset of cases (15-20%), relevant stromal detachment or low leukemia survival led to samples not meeting all quality control standards. Drugs were ranked by the area under the dose response curve (AUC) and referenced to all previous samples. This patient-specific ex vivo drug fingerprint was reported to the treating physician with a typical turn-around time of less than two weeks. Results Dexamethasone sensitivities measured ex vivo in BM samples from pediatric front-line patients, who were treated on a one-week cytoreductive steroid pre-phase followed by a four-drug induction regimen, correlated significantly with clinical responses at d8 (PGR vs. PPR, p<0.001), d15 (flow MRD below 10%, p<0.001) and d33 (PCR MRD below E-2, p<0.001). By integrating dose-response profiles from all patients, we observed differential drug activities across cytogenetic groups and identified exceptional responders to Venetoclax in very high-risk subtypes including TCF3::HLF (5 patients, AUC percent rank: 0.03 - 0.19) as well as refractory T-ALL. In 77 r/r T-ALL patients from 12 different countries, DRP supported selection of a personalized bridging regimen prior to consolidation with cellular therapy. Top-scoring drug sensitivities (10th percentile of AUC) were found for a variety of agents including Imatinib, Dasatinib, Venetoclax, Bortezomib, Selinexor and different nucleoside analogs. Eleven T-ALL and 5 BCP-ALL patients received a DRP-guided bridging element involving compounds with high ex vivo drug efficacy and complemented by chemotherapy (Table 1). The other 66 r/r patients were treated according to physician's choice incorporating information on drug resistance detected by DRP in real time. Bridging strategies implementing DRP-guided therapy blocks stabilized or reduced disease burden in 15/16 patients allowing HSCT or infusion of CAR-T cells in 12 patients with 8 patients still being in remission at last follow-up. Conclusion We describe an array of pharmacological sensitivity and resistance patterns across a heterogeneous, high-risk cohort of BCP- and T-ALL patients. We identified individual susceptibilities to targeted agents of different classes including BH3-mimetics, proteasome, tyrosine kinase and exportin inhibitors as well as standard-of-care chemotherapeutics. Prospective recording of drug response profiles offers opportunities to systematically correlate ex vivo and clinical responses and inform personalized therapies. These data strongly advocate inclusion of DRP into randomized, early and late phase clinical trials for high-risk pediatric ALL, comprehensively integrating molecular and functional precision information to improve patient stratification and long-term outcome after HSCT and CAR-T.

Small Molecule Cocktails Promote Fibroblast-to-Leydig-like Cell Conversion for Hypogonadism Therapy.

Male hypogonadism arises from the inadequate production of testosterone (T) by the testes, primarily due to Leydig cell (LC) dysfunction. Small molecules possess several advantages, including high cell permeability, ease of synthesis, standardization, and low effective concentration. Recent investigations have illuminated the potential of small molecule combinations to facilitate direct lineage reprogramming, removing the need for transgenes by modulating cellular signaling pathways and epigenetic modifications. In this study, we have identified a specific cocktail of small molecules, comprising forskolin, DAPT, purmorphamine, 8-Br-cAMP, 20α-hydroxycholesterol, and SAG, capable of promoting the conversion of fibroblasts into Leydig-like cells (LLCs). These LLCs expressed key genes involved in testosterone synthesis, such as Star, Cyp11a1, and Hsd3b1, and exhibited the ability to secrete testosterone in vitro. Furthermore, they successfully restored serum testosterone levels in testosterone-castrated mice in vivo. The small molecule cocktails also induced alterations in the epigenetic marks, specifically H3K4me3, and enhanced chromosomal accessibility on core steroidogenesis genes. This study presents a reliable methodology for generating Leydig-like seed cells that holds promise as a novel therapeutic approach for hypogonadism.

Dual biological therapy and small molecules in pediatric inflammatory bowel disease

Inflammatory bowel diseases (IBDs) including Crohn’s disease (CD), ulcerative colitis (UC) and inflammatory bowel disease unclassified (IBD-U) are chronic inflammatory disorders which can affect the gastrointestinal tract. Anti-tumor necrosis factors antibodies (anti-TNFα) such as infliximab (IFX) and adalimumab (ADA) are the first line biological therapy for severe or complicated IBDs in pediatric age. Second line therapeutic options as vedolizumab (VDZ) and ustekinumab (UST) are currently used off-label in pediatric age. Furthermore, despite optimization of biologics, a great proportion of patients may fail to respond to biologic agents (up to 30%) or lose response over the time (around 50%) hence these patients may be left without another valid therapeutic option. Consequently, several efforts have been made in the last years in order to develop new drugs and to contrive new therapeutic strategies. Small molecule drugs (SMDs) and combination therapy with either two biologic agents or with a SMD and a biological agent have recently been proposed. Data on safety and efficacy of these new therapeutic options are limited. The objective of the present review is to summarize the most up-to-date available literature in pediatric IBD.

Selinexor With Anti-PD-1 Antibody as a Potentially Effective Regimen for Patients With Natural Killer/T-Cell Lymphoma Failing Prior L-Asparaginase and PD-1 Blockade.

Natural killer/T-cell lymphoma (NKTCL) is a rare and heterogeneous tumor type of non-Hodgkin's lymphoma (NHL) with a poor clinical outcome. There is no standardized salvage treatment failing l-asparaginase-based regimens. Here we report our retrospective results of the combined use of selinexor and PD-1 blockade (tislelizumab) in 5 patients with NKTCL who had exhausted almost all available treatments. A total of 5 patients with relapsed/refractory(R/R) NK/T-cell lymphomas failing prior l-asparaginase and anti-PD-1 antibody were retrospectively collected. They were treated with at least one cycle of XPO1 inhibitor plus the same anti-PD-1 antibody. Anti-PD-1 antibody (Tislelizumab) was administrated at 200 mg on day 1 every 3 weeks and selinexor doses and schedules ranged from 40 mg weekly for 2 weeks per 21-day cycle to 60 mg weekly per cycle. Five patients with relapsed NKTCL with extensive organ involvement including 4 central nervous system (CNS) infiltration patients were included. Four patients achieved objective responses including 3 complete responses (CR) and 1 partial response (PR). After a median follow-up time of 14.5 (range, 5-22) months, 1 patient was still in remission with CR, and the other 4 patients discontinued due to disease progression with a median progression-free survival (PFS) of 6 months and median overall survival (OS) of 12 months. Four patients with CNS involvement achieved a median OS of 8 months. Our data suggest that selinexor in combination with an anti-PD-1 antibody is a promising small molecule and immunotherapy combination regimen for patients with relapsed or refractory NKTCL.

Automated generation of hiPSC-derived hepatic progeny by cost-efficient compounds.

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) hold great promise for liver disease modeling, drug discovery and drug toxicity screens. Yet, several hurdles still need to be overcome, including among others decrease in the cost of goods to generate HLCs and automation of the differentiation process. We here describe that use of an automated liquid handling system results in highly reproducible HLC differentiation from hPSCs. This enabled us to screen 92 chemicals to replace expensive growth factors at each step of the differentiation protocol to reduce the cost of goods of the differentiation protocol by approximately 79%. In addition, we also evaluated several recombinant extracellular matrices (ECM) to replace Matrigel. We demonstrated that differentiation of hPSCs on Laminin-521 using an optimized small molecule combination resulted in HLCs that were transcriptionally identical to HLCs generated using the growth factor combinations. In addition, the HLCs created using the optimized small molecule combination secreted similar amounts of albumin and urea, and relatively low concentrations of alfa-fetoprotein (AFP), displayed similar CYP3A4 functionality and a similar drug toxicity susceptibility as HLCs generated with growth factor cocktails. The broad applicability of the new differentiation protocol was demonstrated for four different hPSC lines. This allowed the creation of a scalable, xeno-free, and cost-efficient hPSC-derived HLC culture, suitable for high throughput disease modeling and drug screenings, or even for the creation of HLCs for regenerative therapies.

A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

Co‐Targeting c‐Myc and Bcl‐2 by Oral Small Molecule Combination of WBC100 and Venetoclax Effectively Controls Acute Myeloid Leukemia in Preclinical Models

AbstractUncontrolled proliferation and apoptosis evasion are two hallmarks of acute myeloid leukemia (AML), but the molecular mechanisms remain poorly understood. In this study, it is demonstrated that the double over‐expresser of oncoprotein c‐Myc and anti‐apoptotic protein Bcl‐2 is a critical “genetic overdrive” of proliferation and apoptosis evasion in AML and is associated with poor genetic alterations. Double‐knockdown of c‐Myc/ Bcl‐2 synergistically kills AML cells in vitro and in vivo. Moreover, a novel oral small molecule combination co‐targeting c‐Myc and Bcl‐2 with WBC100 and Venetoclax (VEN) at low doses are developed. Importantly, the study shows that this combination results in deep and durable remissions of AML, and its efficacy is superior to the frontline combination of Venetoclax and hypomethylation azacitidine (AZA) in AML mouse models and PDX models from relapsed or refractory AML patients. Mechanically, Bcl‐2 knockdown induces mitochondrial outer membrane permeabilization and c‐Myc‐knockdown impairs mitochondria biogenesis. Co‐targeting c‐Myc/Bcl‐2 reciprocally abrogates over‐proliferation and apoptosis resistance via forming a double hit to mitochondrial biogenesis and apoptosis machinery. The findings for the first time demonstrate that co‐targeting c‐Myc/Bcl‐2 by the novel oral small molecule combination of WBC100/Venetoclax is a promising and convenient therapy for AML and support its clinical trial.

Potent suppression of viral rebound after analytical treatment interruption in ART-suppressed SHIV-infected animals by a CD4 binding site bNAb

Abstract Broadly neutralizing antibodies (bNAbs) are promising immunotherapeutic biological agents against HIV-1 that can be used to complement small-molecule combination antiretroviral therapy (cART) drugs for the treatment of HIV-1 infection. There have been several analytical treatment interruption (ATI) studies utilizing bNAbs in ART-suppressed HIV-1-infected patients in which bNAbs are infused during an ART cessation period. To model these clinical studies, bNAbs can be administered to ART-suppressed SHIV-infected rhesus macaques (RM). However, delivery of human bNAbs to RMs induces formation of anti-drug antibodies (ADA) which shorten the half-life of the bNAb and limit the time during which effectiveness can be assessed. In this study, we infused six ART-suppressed SHIV-infected RMs with four 6–10 mg/kg doses of VRC01v23, a next generation CD4-binding site bNAb, every 2–4 weeks for a total duration of 10 weeks. Prior to bNAb treatment, three 50 mg/kg doses of an anti-CD20 antibody were given two weeks apart in order to deplete peripheral B cells and eliminate ADA. Five of six animals had persistent B cell depletion and showed no evidence of ADA after infusions of VRC01v23 and cessation of ART. No viral rebound was observed in these five animals. The sixth animal developed ADA against VRC01v23 resulting in rapid clearance of VRC01v23 from circulation and viral rebound. These data demonstrate that B cell depletion can limit the development of ADA against human bNAbs allowing for the assessment of long-term therapeutic potential of human bNAbs in RMs. Supported by intramural NIAID funding

595 Anticodon engineered transfer ibonucleic acid and small-molecule combination treatment of cystic fibrosis nonsense mutations

595 Anticodon engineered transfer ibonucleic acid and small-molecule combination treatment of cystic fibrosis nonsense mutations

Transient targeting of BIM-dependent adaptive MCL1 preservation enhances tumor response to molecular therapeutics in non-small cell lung cancer.

Despite remarkable efficacy, targeted treatments often yield a subpopulation of residual tumor cells in part due to non-genetic adaptions. Previous mechanistic understanding on the emergence of these drug-tolerant persisters (DTPs) has been limited to epigenetic and transcriptional reprogramming. Here, by comprehensively interrogating therapy-induced early dynamic protein changes in diverse oncogene-addicted non-small cell lung cancer models, we identified adaptive MCL1 increase as a new and universal mechanism to confer apoptotic evasion and DTP formation. In detail, acute MAPK signaling disruption in the presence of genotype-based tyrosine kinase inhibitors (TKIs) prompted mitochondrial accumulation of pro-apoptotic BH3-only protein BIM, which sequestered MCL1 away from MULE-mediated degradation. A small-molecule combination screen uncovered that PI3K-mTOR pathway blockade prohibited MCL1 upregulation. Biochemical and immunocytochemical evidence indicated that mTOR complex 2 (mTORC2) bound and phosphorylated MCL1, facilitating its interaction with BIM. As a result, short-term polytherapy combining antineoplastic TKIs with PI3K, mTOR or MCL1 inhibitors sufficed to prevent DTP development and promote cancer eradication. Collectively, these findings support that upfront and transient targeting of BIM-dependent, mTORC2-regulated adaptive MCL1 preservation holds enormous promise to improve the therapeutic index of molecular targeted agents.

Expert workshop summary: Advancing toward a standardized murine model to evaluate treatments for antimicrobial resistance lung infections.

The rise in antimicrobial resistance (AMR), and increase in treatment-refractory AMR infections, generates an urgent need to accelerate the discovery and development of novel anti-infectives. Preclinical animal models play a crucial role in assessing the efficacy of novel drugs, informing human dosing regimens and progressing drug candidates into the clinic. The Innovative Medicines Initiative-funded “Collaboration for prevention and treatment of MDR bacterial infections” (COMBINE) consortium is establishing a validated and globally harmonized preclinical model to increase reproducibility and more reliably translate results from animals to humans. Toward this goal, in April 2021, COMBINE organized the expert workshop “Advancing toward a standardized murine model to evaluate treatments for AMR lung infections”. This workshop explored the conduct and interpretation of mouse infection models, with presentations on PK/PD and efficacy studies of small molecule antibiotics, combination treatments (β-lactam/β-lactamase inhibitor), bacteriophage therapy, monoclonal antibodies and iron sequestering molecules, with a focus on the major Gram-negative AMR respiratory pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Here we summarize the factors of variability that we identified in murine lung infection models used for antimicrobial efficacy testing, as well as the workshop presentations, panel discussions and the survey results for the harmonization of key experimental parameters. The resulting recommendations for standard design parameters are presented in this document and will provide the basis for the development of a harmonized and bench-marked efficacy studies in preclinical murine pneumonia model.