Trident cold atmospheric plasma blocks three cancer survival pathways to overcome therapy resistance

Therapy resistance is responsible for most cancer-related death and is mediated by the unique ability of cancer cells to leverage metabolic conditions, signaling molecules, redox status, and other pathways for their survival. Interestingly, many cancer survival pathways are susceptible to disturbances in cellular reactive oxygen species (ROS) and may therefore be disrupted by exogenous ROS. Here, we explore whether trident cold atmospheric plasma (Tri-CAP), a gas discharge with exceptionally low-level ROS, could inhibit multiple cancer survival pathways together in a murine cell line model of therapy-resistant chronic myeloid leukemia (CML). We show that Tri-CAP simultaneously disrupts three cancer survival pathways of redox deregulation, glycolysis, and proliferative AKT/mTOR/HIF-1α signaling in this cancer model. Significantly, Tri-CAP blockade induces a very high rate of apoptotic death in CML cell lines and in primary CD34+ hematopoietic stem and progenitor cells from CML patients, both harboring the therapy-resistant T315I mutation. In contrast, nonmalignant controls are minimally affected by Tri-CAP, suggesting it selectively targets resistant cancer cells. We further demonstrate that Tri-CAP elicits similar lethality in human melanoma, breast cancer, and CML cells with disparate, resistant mechanisms and that it both reduces tumor formation in two mouse models and improves survival of tumor-bearing mice. For use in patients, administration of Tri-CAP may be extracorporeal for hematopoietic stem cell transplantation therapy, transdermal, or through its activated solution for infusion therapy. Collectively, our results suggest that Tri-CAP represents a potent strategy for disrupting cancer survival pathways and overcoming therapy resistance in a variety of malignancies.

Targeting PI3K/Akt signal transduction for cancer therapy

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a crucial role in various cellular processes and is aberrantly activated in cancers, contributing to the occurrence and progression of tumors. Examining the upstream and downstream nodes of this pathway could allow full elucidation of its function. Based on accumulating evidence, strategies targeting major components of the pathway might provide new insights for cancer drug discovery. Researchers have explored the use of some inhibitors targeting this pathway to block survival pathways. However, because oncogenic PI3K pathway activation occurs through various mechanisms, the clinical efficacies of these inhibitors are limited. Moreover, pathway activation is accompanied by the development of therapeutic resistance. Therefore, strategies involving pathway inhibitors and other cancer treatments in combination might solve the therapeutic dilemma. In this review, we discuss the roles of the PI3K/Akt pathway in various cancer phenotypes, review the current statuses of different PI3K/Akt inhibitors, and introduce combination therapies consisting of signaling inhibitors and conventional cancer therapies. The information presented herein suggests that cascading inhibitors of the PI3K/Akt signaling pathway, either alone or in combination with other therapies, are the most effective treatment strategy for cancer.

RIPK1 Is Cleaved by 3C Protease of Rhinovirus A and B Strains and Minor and Major Groups

Rhinoviruses (RV), like many other viruses, modulate programmed cell death to their own advantage. The viral protease, 3C has an integral role in the modulation, and we have shown that RVA-16 3C protease cleaves Receptor-interacting protein kinase-1 (RIPK1), a key host factor that modulates various cell death and cell survival pathways. In the current study, we have investigated whether this cleavage is conserved across selected RV strains. RIPK1 was cleaved in cells infected with strains representing diversity across phylogenetic groups (A and B) and receptor usage (major and minor groups). The cleavage was abrogated in the presence of the specific 3C protease inhibitor, Rupintrivir. Interestingly, there appears to be involvement of another protease (maybe 2A protease) in RIPK1 cleavage in strains belonging to genotype B. Our data show that 3C protease from diverse RV strains cleaves RIPK1, highlighting the importance of the cleavage to the RV lifecycle.

Phase 1b/2 Study of Vipor (Venetoclax, Ibrutinib, Prednisone, Obinutuzumab, and Lenalidomide) in Relapsed/Refractory and Untreated Mantle Cell Lymphoma: Safety, Efficacy, and Molecular Analysis

Background: Mantle cell lymphoma (MCL) is a biologically and clinically heterogenous B-cell lymphoma that is generally incurable with standard therapies. Novel targeted agents can disrupt key survival pathways in MCL such as regulation of apoptosis (BCL2: venetoclax), B-cell receptor signaling (BTK: ibrutinib), and NF-κB survival pathways (IRF4/SPIB: lenalidomide). As monotherapy, these agents fail to induce deep responses and doublet/triplet regimens often require continuous or maintenance therapy. ViPOR has been shown to be safe and active in non-MCL NHL pts without significant tumor lysis syndrome (TLS) (Melani et al. Blood. 2020; 136(Supplement 1):44-45). We hypothesized that combining agents that target multiple survival pathways with ViPOR will leverage efficacy and time-limited, cyclic dosing will limit toxicities in MCL. Methods: Relapsed/refractory (R/R) and untreated MCL pts with adequate organ function were eligible. In R/R MCL, a phase I "3+3" design was used to determine the maximum tolerated dose (MTD) of 2 dose-levels of dose-escalated venetoclax (200mg and 400mg) PO D2-14 (starting C2) in combination with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide 15mg PO D1-14. A phase II expansion in untreated MCL was included at the MTD. ViPOR q21d x 6C was given without maintenance or consolidation. All pts were admitted C2 for 12d venetoclax escalation and TLS monitoring. All pts received TLS prophylaxis (ppx) with IVFs and allopurinol as well as PCP and G-CSF ppx. VTE ppx was per investigator discretion. Baseline CT, PET, BM, and tumor biopsy was performed with CT scans after cycles 1, 2, 4, and 6 and PET after cycle 6 or at time of suspected complete response (CR). Surveillance CT was performed q3m for 1y, q4m x 1y, q6m x 1y, then annually x 2y. Plasma for ctDNA was collected at baseline, prior to each treatment cycle, at each follow-up visit, and at disease progression. Results: 11 pts have been enrolled and treated; 9 (82%) R/R in dose-escalation and 2 (18%) untreated in dose-expansion. Median age was 71y (range 57-79) with 73% >65y and 64% male. Low, intermediate, and high-risk MIPI occurred in 18%, 64%, and 18% of pts, respectively. Stage IV disease was seen in 91%, with BM involvement in 73%, extranodal disease in 82%, and both in 64%. Disease bulk >5cm occurred in 45% of pts. Blastoid morphology, Ki-67 >30%, and TP53 IHC >50% occurred in 27%, 36%, and 18% of pts, respectively. Median prior therapies in R/R pts was 3 (range 1-4) with 44% receiving prior BTKi, 11% receiving prior CAR-T, and 78% refractory (i.e., <PR) to last therapy. No pts received prior venetoclax or lenalidomide. No dose-limiting toxicities (DLTs) occurred in 9 evaluable pts in the dose-escalation cohort; thus, venetoclax 400mg was used in expansion. G3-4 heme AEs (% cycles) included neutropenia (13%), anemia (11%), and thrombocytopenia (9%). No cases of febrile neutropenia occurred across 46 total cycles. G3-4 non-heme AEs (% pts) included hypokalemia in 3 (33%) pts as well as fatigue, hypomagnesemia, elevated bilirubin, atrial fibrillation, lung infection, and syncope in 1 (11%) pt each. No laboratory or clinical TLS occurred. Dose reductions and delays occurred in 5% and 15% of cycles, respectively. Of 11 pts enrolled, 10 are evaluable for response (1 pt has not yet been restaged) with an overall response rate (ORR) and CR rate of 100% (10/10) and 80% (8/10), respectively (Fig 1A). Of 8 pts who have completed therapy, all 8 (100%) have achieved CR, including all 4 post-BTKi pts, 1 post-CAR-T pt, and 6 refractory pts. With a median potential f/u of 5.2m, median TTR and DOR was 0.7m and not reached, respectively, with 9 (90%) responses ongoing ranging from 0.3m to 14.5m after first response (Fig 1B). One pt with R/R blastoid MCL relapsed in the CNS 9.9m after initial response. Median PFS and OS were both not reached with 10 (91%) pts alive and progression-free and 1 relapse and death from progression at 11.9m. Conclusion: ViPOR is safe in MCL without significant TLS or DLTs using a 12d venetoclax ramp-up on C2 and venetoclax 400mg was taken forward in expansion. Most common G3-4 AEs were hematologic with no febrile neutropenia observed when given with G-CSF ppx. High preliminary activity is noted in MCL pts with fixed-duration ViPOR x 6C, including CRs in refractory, post-BTKi, and post-CAR-T pts. Molecular and ctDNA analyses are ongoing and will be presented at the meeting. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Off-label use of ViPOR in relapsed/refractory and untreated MCL.

Deep Profiling of Chronic Lymphocytic Leukaemia (CLL) and Healthy Immune Cells By Mass Cytometry Resolves Impacts of Venetoclax Pressure

Introduction The BCL-2 inhibitor, venetoclax, is an effective treatment for chronic lymphocytic leukaemia (CLL). Most CLL patients (pts) treated with venetoclax respond, however in the relapsed/refractory (r/r) setting 30-50% patients progress after 2-3 years despite continuous treatment. Although the molecular drivers of relapse are becoming clearer, precisely how the pressure exerted by venetoclax is manifest at the cellular level to engender resistance remains unclear. In this study, we performed mass cytometric (CyTOF) profiling of CLL samples taken from pts during venetoclax dose escalation and employed in vivo modelling to determine the impact of short-term treatment upon survival pathways, proliferation and homing characteristics in both leukemic and normal immune cells. Methods We conducted serial analysis of peripheral blood (PB) samples from 20 pts with r/r CLL (median 2 lines therapy, range 1-5) at baseline and during venetoclax dose escalation (weekly increases of 20, 50, 100, 200, 400 mg) using deep profiling by CyTOF. We measured changes in 43 regulators of immune cells, cell death, proliferation, cell signalling and cancer-related pathways at the single-cell resolution. The data were normalized using CytofRUV, then analyzed using high-dimensional FlowSOM clustering, tSNE/UMAP visualization tools and conventional cytometric analysis. In order to distinguish cell intrinsic and extrinsic impacts, we assayed venetoclax pressure on immune cells with in vivo models using wild-type, Bak -/-Bax Δcd23 and Vav-BCL-2 transgenic mice and haematopoietic chimeras. Results CyTOF analysis of CLL patients demonstrated inter-patient and intra-patient heterogeneity in leukemic cell populations. After six weeks of short-term venetoclax therapy, all pts showed steep decreases in CLL burden in the PB. In the remaining CLL cells, there was a striking dose-dependent increase in amounts of BCL-2, but not MCL-1 and BCL-XL protein expression. The BCL-2 +++ CLL cells remaining after venetoclax therapy were enriched for the proliferative CXCR4 high CD5 low CLL cells. As PB CLL cells are reduced upon venetoclax treatment, there were proportional increases in healthy PB T cells in CLL patients. Compared to baseline samples, all T cell subpopulations were maintained and targets of immune checkpoint inhibitors, such as PD-1, remained unchanged after venetoclax therapy. We then modelled venetoclax responses in vivo by treating mice with 100 mg/kg venetoclax daily for 7 days and assessed the sensitivity of various immune cell subsets, their proliferation and expression of cell survival proteins. Immature lymphocytes were relatively insensitive to venetoclax, while mature naïve populations were depleted. In accord with the CLL patient data, the remaining mature B and T cells expressed very high levels of BCL-2 and were highly proliferative. Lymphocytes lacking the apoptotic effectors BAX and BAK from Bak-/-BaxΔcd23 micedid not exhibit these changes. By contrast, transgenic mice with overexpression of BCL-2 (vav-bcl-2 mice) did not rescue cells from the impacts of venetoclax. These data reveal direct and consistent impacts of venetoclax pressure in normal and leukemic cells at the single cell level. Conclusions Collectively, our findings reveal that short-term treatment with venetoclax exerts pressure on both CLL and healthy immune cells (Figure). Striking changes in the wiring of survival pathways in CLL cells occurred shortly after venetoclax treatment, suggesting that therapies targeting orthogonal survival pathways should be considered shortly after dose-escalation. Furthermore, the limited changes in healthy T cells opens a window for targeting these cells by adjunct immune checkpoint inhibitors to achieve deeper responses. Figure 1 Figure 1. Disclosures Teh: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Tan: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Trussart: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Luo: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Thijssen: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Roberts: Janssen: Research Funding; Abbvie: Research Funding; The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax; Servier: Research Funding. Huang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Speed: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Anderson: The Walter and Eliza Hall Institute: Honoraria, Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax, Speakers Bureau. Gray: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax.

miRNA biogenesis, function and their therapeutic implications

Research in microRNA has witnessed a significant breakthrough as it is significantly involved in numerous biological processes. MicroRNAs are short (~22 nucleotides) non-coding RNAs, are cell/ tissuespecifically expressed and are temporarily regulated. They regulate post-transcription output of proteins by targeting mRNAs for translational repression or degradation. This review is written with searches related to current trends and application of miRNAs from databases of Pubmed and Google. microRNAs are known to participate in developmental and physiological processes and their dysregulation contributes to various disease progression. The benefit of using microRNA approaches depends on its ability to simultaneously target multiple effectors of cell differentiation, proliferation and survival pathways. Dysfunctional miRNAs have been implicated in the development of various disorders. These small RNA molecules serve as post-transcriptional regulators of gene expression and are considered as reliable clinical biomarkers. Considering its tissue and disease-specific expression involving pathophysiological processes, microRNAs are proposed as useful biomarker for prognosis, assessing disease risk. microRNAs can serve as potent biomarker for cancer and other diseases such as neurodegenerative diseases, cardiovascular diseases and diabetes. This review mainly focuses on emphasizing the biology, dysregulation associated with various disease models and use of microRNAs as potential biomarkers for disease diagnosis.

SENP3 Promotes an Mff-Primed Bcl-xL-Drp1 Interaction Involved in Cell Death Following Ischemia

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-xL, an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-xL interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-xL interaction. Our data suggest that Mff primes Drp1 binding to Bcl-xL at the mitochondria and that Mff and Bcl-xL can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-xL interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-xL interaction. Expressing a Bcl-xL mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-xL interaction that contributes to cell death following ischemia.

A highly annotated database of genes associated with platinum resistance in cancer

Platinum-based chemotherapy, including cisplatin, carboplatin, and oxaliplatin, is prescribed to 10-20% of all cancer patients. Unfortunately, platinum resistance develops in a significant number of patients and is a determinant of clinical outcome. Extensive research has been conducted to understand and overcome platinum resistance, and mechanisms of resistance can be categorized into several broad biological processes, including (1) regulation of drug entry, exit, accumulation, sequestration, and detoxification, (2) enhanced repair and tolerance of platinum-induced DNA damage, (3) alterations in cell survival pathways, (4) alterations in pleiotropic processes and pathways, and (5) changes in the tumor microenvironment. As a resource to the cancer research community, we provide a comprehensive overview accompanied by a manually curated database of the >900 genes/proteins that have been associated with platinum resistance over the last 30 years of literature. The database is annotated with possible pathways through which the curated genes are related to platinum resistance, types of evidence, and hyperlinks to literature sources. The searchable, downloadable database is available online at http://ptrc-ddr.cptac-data-view.org.

Regulation of STAT3 and its role in cardioprotection by conditioning: focus on non-genomic roles targeting mitochondrial function

Ischemia–reperfusion injury (IRI) is one of the biggest challenges for cardiovascular researchers given the huge death toll caused by myocardial ischemic disease. Cardioprotective conditioning strategies, namely pre- and post-conditioning maneuvers, represent the most important strategies for stimulating pro-survival pathways essential to preserve cardiac health. Conditioning maneuvers have proved to be fundamental for the knowledge of the molecular basis of both IRI and cardioprotection. Among this evidence, the importance of signal transducer and activator of transcription 3 (STAT3) emerged. STAT3 is not only a transcription factor but also exhibits non-genomic pro-survival functions preserving mitochondrial function from IRI. Indeed, STAT3 is emerging as an influencer of mitochondrial function to explain the cardioprotection phenomena. Studying cardioprotection, STAT3 proved to be crucial as an element of the survivor activating factor enhancement (SAFE) pathway, which converges on mitochondria and influences their function by cross-talking with other cardioprotective pathways. Clearly there are still some functional properties of STAT3 to be discovered. Therefore, in this review, we highlight the evidence that places STAT3 as a promoter of the metabolic network. In particular, we focus on the possible interactions of STAT3 with processes aimed at maintaining mitochondrial functions, including the regulation of the electron transport chain, the production of reactive oxygen species, the homeostasis of Ca2+ and the inhibition of opening of mitochondrial permeability transition pore. Then we consider the role of STAT3 and the parallels between STA3/STAT5 in cardioprotection by conditioning, giving emphasis to the human heart and confounders.

Lamin B1 Accumulation’s Effects on Autosomal Dominant Leukodystrophy (ADLD): Induction of Reactivity in the Astrocytes

Autosomal dominant leukodystrophy (ADLD) is an extremely rare and fatal neurodegenerative disease due to the overexpression of the nuclear lamina component Lamin B1. Many aspects of the pathology still remain unrevealed. This work highlights the effect of Lamin B1 accumulation on different cellular functions in an ADLD astrocytic in vitro model. Lamin B1 overexpression induces alterations in cell survival signaling pathways with GSK3β inactivation, but not the upregulation of β-catenin targets, therefore resulting in a reduction in astrocyte survival. Moreover, Lamin B1 build up affects proliferation and cell cycle progression with an increase of PPARγ and p27 and a decrease of Cyclin D1. These events are also associated to a reduction in cell viability and an induction of apoptosis. Interestingly, ADLD astrocytes trigger a tentative activation of survival pathways that are ineffective. Finally, astrocytes overexpressing Lamin B1 show increased immunoreactivity for both GFAP and vimentin together with NF-kB phosphorylation and c-Fos increase, suggesting astrocytes reactivity and substantial cellular activation. These data demonstrate that Lamin B1 accumulation is correlated to biochemical, metabolic, and morphologic remodeling, probably related to the induction of a reactive astrocytes phenotype that could be strictly associated to ADLD pathological mechanisms.