Pro-survival Molecules Research Articles

Abstract A015: Novel genetic driver for the genesis of muscle invasion in bladder cancer

Abstract Non-muscle invasive bladder cancer (NMIBC) is a heterogeneous type of urothelial carcinoma with a substantial risk (>50%) of tumor progression and muscle invasion. Our sequential studies uniquely unveiled the availability of Retinal Degeneration Protein 3 (RD3) in human adult/fetal tissues and recognized its functional significance in tumor evolution. Herein, we investigated the relevance of RD3 in coordinating de novo MIBC from NMIBC. Compared to the low-grade tumor, RD3 is significantly lost (mRNA, qPCR; protein, ELISA) in grade III and IV patient derived NMIBC cells. In a cohort of (n=55) NMIBC patients, RD3-loss associated with increased tumor invasive potential (complete loss in highly invasive T4), metastasis (vs. primary tumors), therapy pressure (insignificant levels in progressive disease that defies therapy), disease recurrence, and death (custom archived TMA with automated RD3 IHC).Crucially, we observed a significant low-is-worse association to overall survival (p=7.30E-03, hazard ratio (HR) of 5.161), relapse-free survival (p=4.464E-02, HR = 2.328), and progression-free survival (p=7.00E-0.4, HR = 3.264). Further, radio-resistant cells exposed to fractionated irradiation (FIR, 2Gy/Day for 5 days) to a total dose of 10 or 20Gy displayed a dose dependent loss of RD3 recognizing an ongoing acquisition of RD3-loss with therapy pressure. More interestingly, Grade I or III bystander cells (BSE) co-cultured with FIR-exposed cells displayed anew acquisition of RD3-loss and displayed tumor grade-dependent increase (vs. mock-irradiated) in NFkB transcriptional activity (EMSA) and the activation of NFkB trans-activated pro-survival molecules (QPCR profiling of 89 NFkB targets). Together, these results clearly depict that RD3 regulates NMIBC disease pathogenesis, dissemination, acquired loss of RD3 with therapy pressure and further implies its role in the evolution of de novo MIBC from NMIBC. Funding: DOD CA-210339, OCAST-HR19-045, NIH-P20GM103639, NIH-NCI-P30CA225520 and TSET-R23-03. Citation Format: Sreenidhi Mohanvelu, Poorvi Subramanian, Sabir Salim, Dinesh Babu Somasundaram, Sheeja Aravindan, Natarajan Aravindan. Novel genetic driver for the genesis of muscle invasion in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A015.

A high efficiency precision genome editing method with CRISPR in iPSCs

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines.

Abstract 690: Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma

Abstract Glioblastoma (GBM) is a highly aggressive primary brain tumor. Treatment consists of maximal safe tumor resection, chemotherapy (temozolomide) and radiotherapy; however, this aggressive treatment offers only a modest improvement in outcomes, with average life expectancy of approximately 12 months. Consequently, novel, effective and easily translatable therapies are urgently required. While radiotherapy is an effective treatment for many solid tumors, GBM displays an innate radio-resistance that contributes to treatment failure. Therefore, there is a need to identify chemotherapeutics that can be used as radiosensitizers to increase efficacy. Notably, a key pathway involved in therapeutic resistance is apoptotic evasion, which is driven by upregulation of pro-survival BCL-2 proteins that inhibit the pro-apoptotic proteins necessary for the apoptotic response. Recent advances in drug development have produced a range of BH3 mimetics - novel chemotherapeutics with potential to reinstate apoptosis by targeting specific pro-survival molecules. We hypothesized that the radiation sensitivity of GBM cells would be enhanced by using BH3 mimetics to inhibit pro-survival molecules and release the apoptotic block. A panel of BH3 mimetics was tested in combination with radiotherapy across three patient-derived GBM cell lines (G7, E2 and R15), using a 3D clonogenic culture system that recapitulates in vivo growth patterns and responses. We found that the BCL-XL inhibitor, A1331852, significantly radiosensitized all 3 GBM lines. In addition, Western blot analysis revealed A1331852 to induce PARP1 cleavage in G7 cells when used as a single agent. Subsequent in vivo studies were conducted in our well characterized orthotopic G7 xenograft murine model of GBM. Immunohistochemical studies showed that the pro-survival proteins MCL-1 and BCL-XL were upregulated in irradiated tumors. Furthermore, a pharmacokinetic study in the same orthotopic murine model, allowing for drug delivery evaluation, and optimized treatment scheduling. Importantly, A1331852 was found to be blood brain barrier penetrant at 25 mg/kg, with increased drug detected in tumor vs normal brain tissue. From these data, a survival study investigating A1331852 as a radiosensitizing agent was carried out in the same G7 GBM orthotopic xenograft model. Kaplan-Meier analysis showed an increase in survival of mice treated with A133852 plus radiotherapy compared with the radiotherapy only group (p = 0.0309). Overall, we have demonstrated that dependence on BCL-2 family proteins is a vulnerability that can be targeted to improve radiotherapy response in GBM. Furthermore, we have identified a promising BH3 mimetic, A1331852, to use in combination with RT and have completed a proof-of-concept in vivo study that confirms the potential of BH3 mimetics to improve outcomes for patients with GBM. Citation Format: Louise R. Dutton, Clara Mullen, Anna L. Koessinger, Mark Jackson, Katrina Stevenson, Anthony J. Chalmers, Stephen Tait, Kirsteen J. Campbell, Joanna L. Birch. Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma [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 690.

Proteasome Inhibitors against Glioblastoma-Overview of Molecular Mechanisms of Cytotoxicity, Progress in Clinical Trials, and Perspective for Use in Personalized Medicine.

Proteasome inhibitors are moieties targeting the proteolytic activity of a proteasome, with demonstrated efficacy in certain hematological malignancies and candidate drugs in other types of cancer, including glioblastoma (GBM). They disturb the levels of proteasome-regulated proteins and lead to the cell cycle inhibition and apoptosis of GBM cells. The accumulation of cell cycle inhibitors p21 and p27, and decreased levels of prosurvival molecules NFKB, survivin, and MGMT, underlie proteasome inhibitors' cytotoxicity when used alone or in combination with the anti-GBM cytostatic drug temozolomide (TMZ). The evidence gathered in preclinical studies substantiated the design of clinical trials that employed the two most promising proteasome inhibitors, bortezomib and marizomib. The drug safety profile, maximum tolerated dose, and interaction with other drugs were initially evaluated, mainly in recurrent GBM patients. A phase III study on newly diagnosed GBM patients who received marizomib as an adjuvant to the Stupp protocol was designed and completed in 2021, with the Stupp protocol receiving patients as a parallel control arm. The data from this phase III study indicate that marizomib does not improve the PFS and OS of GBM patients; however, further analysis of the genetic and epigenetic background of each patient tumor may shed some light on the sensitivity of individual patients to proteasome inhibition. The mutational and epigenetic makeup of GBM cells, like genetic alterations to TP53 and PTEN, or MGMT promoter methylation levels may actually determine the response to proteasome inhibition.

Riluzole Suppresses Growth and Enhances Response to Endocrine Therapy in ER+ Breast Cancer.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.

Multifunctional cytokine production marks influenza A virus-specific CD4 T cells with high expression of survival molecules.

Cytokine production by memory T cells is a key mechanism of T cell mediated protection. However, we have limited understanding of the persistence of cytokine producing T cells during memory cell maintenance and secondary responses. We interrogated antigen-specific CD4 T cells using a mouse influenza A virus infection model. Although CD4 T cells detected using MHCII tetramers declined in lymphoid and non-lymphoid organs, we found similar numbers of cytokine+ CD4 T cells at days 9 and 30 in the lymphoid organs. CD4 T cells with the capacity to produce cytokines expressed higher levels of pro-survival molecules, CD127 and Bcl2, than non-cytokine+ cells. Transcriptomic analysis revealed a heterogeneous population of memory CD4 T cells with three clusters of cytokine+ cells. These clusters match flow cytometry data and reveal an enhanced survival signature in cells capable of producing multiple cytokines. Following re-infection, multifunctional T cells expressed low levels of the proliferation marker, Ki67, whereas cells that only produce the anti-viral cytokine, interferon-γ, were more likely to be Ki67+ . Despite this, multifunctional memory T cells formed a substantial fraction of the secondary memory pool. Together these data indicate that survival rather than proliferation may dictate which populations persist within the memory pool.

The RNA binding proteins TIA1 and TIAL1 promote Mcl1 mRNA translation to protect germinal center responses from apoptosis

Germinal centers (GCs) are essential for the establishment of long-lasting antibody responses. GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into functional changes in the cell proteome. However, the critical proteins driving these key mechanisms are still unknown. Here, we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses. TIA1- and TIAL1-deficient GC B cells fail to undergo antigen-mediated positive selection, expansion and differentiation into B-cell clones producing high-affinity antibodies. Mechanistically, TIA1 and TIAL1 control the transcriptional identity of dark- and light-zone GC B cells and enable timely expression of the prosurvival molecule MCL1. Thus, we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.

NFE2L2 and STAT3 Converge on Common Targets to Promote Survival of Primary Lymphoma Cells.

NFE2L2 and STAT3 are key pro-survival molecules, and thus, their targeting may represent a promising anti-cancer strategy. In this study, we found that a positive feedback loop occurred between them and provided evidence that their concomitant inhibition efficiently impaired the survival of PEL cells, a rare, aggressive B cell lymphoma associated with the gammaherpesvirus KSHV and often also EBV. At the molecular level, we found that NFE2L2 and STAT3 converged in the regulation of several pro-survival molecules and in the activation of processes essential for the adaption of lymphoma cells to stress. Among those, STAT3 and NFE2L2 promoted the activation of pathways such as MAPK3/1 and MTOR that positively regulate protein synthesis, sustained the antioxidant response, expression of molecules such as MYC, BIRC5, CCND1, and HSP, and allowed DDR execution. The findings of this study suggest that the concomitant inhibition of NFE2L2 and STAT3 may be considered a therapeutic option for the treatment of this lymphoma that poorly responds to chemotherapies.

Bacterial blockade of host protein synthesis triggers cell death in dendritic cells to restrict Legionella pneumophilainfection

Abstract Legionella pneumophila is a facultative intracellular bacterial pathogen that is responsible for a pneumonia called Legionnaire’s disease. Legionella invades cells by using a type IV secretion system that delivers effector proteins into the host cell cytosol to facilitate its intracellular survival and replication. Macrophages serve as the primary replicative niche for Legionella. In contrast, dendritic cells (DCs) have been shown to rapidly restrict Legionella infection by activating cell death. The host and bacterial factors that induce this response remain elusive. We found that DCs undergo caspase-dependent cell death and that this response is triggered by a subset of secreted effectors that block host protein synthesis. Specifically, we show that these effectors decrease the levels of pro-survival proteins in DCs, thus leaving cells poised to undergo cell death. Altogether, our data show that pro-survival molecules in DCs serve as guard proteins that detect pathogenic activities to limit bacterial replication. Supported by a National Science Foundation Graduate Research Fellowship Program (DGE-2236662)

Targeted tumor killing by pomegranate polyphenols: Pro-oxidant role of a classical antioxidant

One of the key biochemical features that distinguish a cancer cell from normal cells is its persistent pro-oxidative state that leads to intrinsic oxidative stress. Malignant cells have evolved sophisticated adaptation systems that involve high dependency on antioxidant functions and upregulation of pro-survival molecules to counteract the deleterious effects of reactive species and to maintain dynamic redox balance. This situation renders them vulnerable to further oxidative challenges by exogenous agents. In the present study, we advocated that pomegranate polyphenols act as pro-oxidants and trigger ROS-mediated apoptosis in cancer cells. With the help of both in vitro and in vivo models, we have established that pomegranate fruit extract (PFE) can cause a significant reduction in tumor proliferation while leaving normal tissues and cells unharmed. Administration of PFE (0.2% v/v) in Erhlich's ascites carcinoma-bearing mice for 3 weeks, inhibited the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element signaling cascade, increased intracellular reactive oxygen species content, altered glutathione cycle thereby activating reactive oxygen species-induced apoptotic pathway in Erhlich's ascites carcinoma cells. Moreover, PFE mitigated epithelial to mesenchymal transition and migration in triple negative breast cancer cells (MDA-MB 231 cells) by down-regulating nuclear factor kappa light-chain-enhancer of activated B cells. Pre-treatment of tumor cells with N-acetyl cysteine protected these cells from undergoing PFE-induced apoptosis while siRNA-mediated silencing of Nuclear factor (erythroid-derived 2)-like 2 and nuclear factor kappa light-chain-enhancer of activated B cells in tumor cells increased the cytotoxic potential and pro-oxidative activity of PFE, indicating a clear role of these transcription factors in orchestrating the anticancer/pro-oxidative properties of PFE. The seminal findings provided may be exploited to develop potential therapeutic targets for selective killing of malignant cells.

Complement component 3 protects human bronchial epithelial cells from cigarette smoke-induced oxidative stress and prevents incessant apoptosis.

The complement component 3 (C3) is a pivotal element of the complement system and plays an important role in innate immunity. A previous study showed that intracellular C3 was upregulated in airway epithelial cells (AECs) from individuals with end-stage chronic obstructive pulmonary disease (COPD). Accumulating evidence has shown that cigarette smoke extract (CSE) induces oxidative stress and apoptosis in AECs. Therefore, we investigated whether C3 modulated cigarette smoke-induced oxidative stress and apoptosis in AECs and participated in the pathogenesis of COPD. We found increased C3 expression, together with increased oxidative stress and apoptosis, in a cigarette smoke-induced mouse model of COPD and in AECs from patients with COPD. Different concentrations of CSEinduced C3 expression in 16HBE cells in vitro. Interestingly, C3 knockdown (KD) exacerbated oxidative stress and apoptosis in 16HBE cells exposed to CSE. Furthermore, C3 exerted its pro-survival effects through JNK inhibition, while exogenous C3 partially rescued CSE-induced cell death and oxidative stress in C3 KD cells. These data indicate that locally produced C3 is an important pro-survival molecule in AECs under cigarette smoke exposure, revealing a potentially novel mechanism in the pathogenesis of COPD.

Clearance of HIV-1 or SIV reservoirs by promotion of apoptosis and inhibition of autophagy: Targeting intracellular molecules in cure-directed strategies.

The reservoirs of the HIV display cellular properties resembling long-lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long-lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV-1 by selective elimination of host cells harboring replication-competent HIV (SECH). While reactivation of HIV-1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up-regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV-1 LTR-directed gene expression, such as NF-κB, AP-1, and Hif-1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl-xL, Mcl-1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV-1 proteins. SECH treatments cleared HIV-1 infections in humanized mice in vivo and in HIV-1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Bclaf1 regulates c-FLIP expression and protects cells from TNF-induced apoptosis and tissue injury.

TNF stimulation generates pro-survival signals through activation of NF-κB that restrict the build-in death signaling triggered by TNF. The competition between TNF-induced survival and death signals ultimately determines the fate of a cell. Here, we report the identification of Bclaf1 as a novel component of the anti-apoptotic program of TNF. Bclaf1 depletion in multiple cells sensitizes cells to TNF-induced apoptosis but not to necroptosis. Bclaf1 exerts its anti-apoptotic function by promoting the transcription of CFLAR, a caspase 8 antagonist, downstream of NF-κB activation. Bclaf1 binds to the p50 subunit of NF-κB, which is required for Bclaf1 to stimulate CFLAR transcription. Finally, in Bclaf1 siRNA administered mice, TNF-induced small intestine injury is much more severe than in control mice with aggravated signs of apoptosis and pyroptosis. These results suggest Bclaf1 is a key regulator in TNF-induced apoptosis, both invitro and invivo.

Expression of the purinergic receptor P2RX7 marks early effector CD8+ T cells with increased potential for both long-lived circulating and resident memory cell generation

Abstract Memory CD8+ T cells protect against secondary viral infections. They develop and maintain exclusively in circulation (e.g. central memory - Tcm) or are excluded from re-circulation (resident memory - Trm). Circulating memory CD8+ T cells arises from memory precursors (MPs) which express memory, pro-survival, and pluripotency molecules. Trm generation, in contrast, is still not fully understood. Previous reports suggest that Trm and Tcm have a common precursor present at the early effector phase; yet, the nature of this precursor is elusive. We have found that the extracellular ATP receptor P2RX7 promotes both Tcm and Trm generation. Moreover, P2RX7 is quickly upregulated in some (but not all) early effectors and early P2RX7 ablation hinders both Tcm and Trm generation. Thus, we postulated that P2RX7 in early effectors may mark a Tcm/Trm common precursor. Comparison between high (P2RX7hi) and low (P2RX7lo) P2RX7-expressing CD8+ T cells shows a correlation between memory markers and P2RX7, with CD62L+ Tcm precursors exclusively found in P2RX7hi cells. RNA-seq of P2RX7hi and P2RX7lo early effectors show survival, memory and pluripotency genes and pathways are upregulated in P2RX7hi cells. Conversely, many terminal effector and apoptosis genes are upregulated in P2RX7lo cells. P2RX7lo early effectors and MPs have increased apoptosis. Upon transfer into infection-matched mice, P2RX7hi MPs preferentially survive and form Tcm compared to P2RX7lo MPs. This is even more apparent when P2RX7hi and P2RX7lo early effectors are compared: approximately 100x more P2RX7hi cells form memory when compared to P2RX7lo cells, both Tcm and Trm. Our data suggest that P2RX7 in early effector CD8+ T cells promotes the generation of a common Tcm/Trm precursor.

Biosynthesis of silver nanoparticles using Sambucus nigra L. fruit extract for targeting cell death in oral dysplastic cells

The study aims to evaluate the impact of silver nanoparticles, phytosynthesized with polyphenols from Sambucus nigra L. (SN) fruit extract (AgSN), on dysplastic oral keratinocytes (DOK) and human gingival fibroblasts (HGF) in terms of cell viability and apoptosis. The morphology and ultrastructure of treated cells as well as the mechanisms involved in cell death induction were investigated in DOK cultures.The structure of AgSN was studied by using the appropriate analysis tools such as UV–Vis, transmission electron microscopy, Raman spectroscopy, dynamic light scattering (DLS) and zeta potential assessment. DOK and HGF were treated either with silver nanoparticles capped with Sambucus nigra L. extract or with SN extract. Untreated cells were used as controls. Viability was determined by MTS assay. Transmission electronic microscopy (TEM) was used to evaluate the intracellular localization of the nanoparticles at 4 and 24 h. Annexin V-FITC/propidium iodide staining and the expressions of p53, BAX, BCL2, NFkB, phosphorylated NFkB (pNFkB), pan AKT, pan phosphoAKT, LC3B and ɣH2AX were evaluated to quantify the cell death. ELISA measurements of TNF-α and TRAIL was used for the study of the inflammatory response. Oxidative stress damage induced by nanoparticles was assessed by the malondialdehyde (MDA) level. Silver nanoparticles stimulated HGF proliferation and significantly diminished DOK viability at doses higher than 20 μg/ml. TEM analysis demonstrated the internalization of silver nanoparticles and showed ultrastructural changes of cells such as the appearance of vacuoles, autophagosomes, endosomes. AgSN inhibited the pro-survival molecules and regulators of apoptosis, diminished oxidative stress and inflammation and induced cell death through various mechanisms: necrosis, autophagy and DNA lesions. SN extract had antioxidant and anti-inflammatory effect and increased the DNA lesions and autophagy in DOK cells. Silver nanoparticles protected the normal cells and induced cell death in dysplastic cells by different mechanisms thus offering beneficial effects in the treatment of oral dysplasia.

Co-Chaperone Bag-1 Plays a Role in the Autophagy-Dependent Cell Survival through Beclin 1 Interaction

Expression levels of the major mammalian autophagy regulator Beclin 1 and its interaction with Bcl-2 regulate the switch between autophagic cell survival and apoptotic cell death pathways. However, some of the regulators and the precise mechanisms of these processes still remain elusive. Bag-1 (Bcl-2 associated athanogene-1), a member of BAG family proteins, is a multifunctional pro-survival molecule that possesses critical functions in vital cellular pathways. Herein, we report the role of Bag-1 on Bcl-2/Beclin 1 crosstalk through indirectly interacting with Beclin 1. Pull-down experiments suggested a molecular interaction between Bag-1 and Beclin 1 in breast cancer cell lines. On the other hand, in vitro binding assays showed that Bag-1/Beclin 1 interaction does not occur directly but occurs through a mediator molecule. Bag-1 interaction with p-Beclin 1 (T119), indicator of early autophagy, is increased during nutrient starvation suggesting involvement of Bag-1 in the autophagic regulation. Furthermore, CRISPR/Cas9-mediated Bag-1 knock-out in MCF-7 cells hampered cell survival and proliferation and resulted in decreased levels of total LC3 under starvation. Collectively, we suggest that Bag-1 modulates cell survival/death decision through maintaining macroautophagy as a component of Beclin 1-associated complexes.

The PKN1- TRAF1 signaling axis as a potential new target for chronic lymphocytic leukemia

ABSTRACT TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.

Analysis of cellular models of clonal evolution reveals co-evolution of imatinib and HSP90 inhibitor resistances

Treatment relapse due to clonal evolution was shown to be an independent factor for poor prognosis in advanced stages of chronic myeloid leukemia. Overcoming secondary resistance arising due to clonal evolution is still an unmet need and lack of adequate pre-clinical models hampers the identification of underlying mechanisms and testing of alternate treatment strategies. The current study thus aimed to create cellular models to study molecular mechanisms underlying clonal evolution and identify strategies to overcome the secondary drug resistance. Analysis of cell lines derived from three independent cell-based screens revealed the co-evolution specifically of imatinib and HSP90 inhibitor (HSP90i) resistances despite their exposure to a single inhibitor alone. Molecular and biochemical characterization of these cell lines revealed additional cytogenetic abnormalities, differential activation of pro-survival signaling molecules and over expression of ABL kinase and HSP90 genes. Importantly, all the imatinib-HSP90i dual resistant cell lines remained sensitive to sorafenib and vorinostat suggesting their utility in treating patients who relapse upon imatinib treatment due to clonal evolution. In addition, we cite similar examples of dual resistance towards various kinase inhibitors and HSP90i in some cell lines that represent solid cancers suggesting co-evolution leading to secondary drug resistance as a pan-cancer phenomenon. Taken together, our results suggest the efficacy of HSP90i in overcoming drug resistance caused by point mutations in the target kinase but not in cases of clonal evolution.

AML-044: LGALS1 Acts as a Pro-Survival Molecule in AML

Context Galectin 1 (LGALS1) participates in diverse survival pathways that support many pro-tumor molecules, especially those regulated by RAS. LGALS1 regulates hematopoietic cell differentiation, so it is not surprising that LGALS1 plays a role in many hematologic malignancies such as acute lymphoblastic leukemia (ALL). However, LGALS1's role in acute myeloid leukemia (AML) is not well defined. Objective We previously found suppression of LGALS1 in AML cell lines sensitized cells to BCL2 inhibitor ABT-737. In this study, we examine the role of LGALS1 in AML. We used an in vivo murine OCI-AML3 xenograft model with control and LGALS1 shRNA cells to test if reduction of LGALS1 affects survival. Gene expression profiling using RNASeq was performed using control and LGALS1 shRNA of p53 WT OCI-AML3 and p53 null THP-1 cells. Patients Analysis of mRNA expression by RNA Seq in AML patients was performed on TCGA AML cohort from NEJM 2013 using cBioPortal. Results Mice with OCI-AML3 cells with LGALS1 shRNA survived significantly longer than control group. There was no difference in leukemia burden in blood but mice bearing OCI-AML3 cells with LGALS1 shRNA showed significant reduction of cells infiltrating the spleen. RNASeq data from THP-1 and OCI-AML3 control and LGALS1 shRNA transductants reveal distinct differences between the two cell lines in number of genes affected, pathways affected, expression of oncogenes, and in transcription factors involved. The p53 pathway is prominent in OCI-AML3 cells while focal adhesion kinase pathway is prominent in THP-1 cells. Of the few genes common to both cell lines, asparagine regulators were reduced. Asparaginase is a common therapy in ALL, so LGALS1 regulation of asparagine metabolism may be important in AML. Examination of LGALS1 mRNA in an AML patient population reveals elevated LGALS1 mRNA is associated with shorter disease-free survival and increased blasts in bone marrow. This data with the xenograft model data suggest LGALS1 may be important in the AML microenvironment. Conclusions Data presented here suggest LGALS1 plays important roles in diverse survival pathways, including p53 pathway, and LGALS1 regulates mobilization of leukemic cells from the leukemia niche. Therefore, a strategy targeting LGALS1 may benefit AML patients.