First person – Nadine Pollak

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Nadine Pollak is first author on ‘ Cell cycle progression and transmitotic apoptosis resistance promote escape from extrinsic apoptosis’, published in JCS. Nadine conducted the research described in this article while a postdoc at the Institute of Cell Biology and Immunology, University of Stuttgart, Germany, initially under the supervision of Prof. Peter Scheurich and subsequently in the lab of Prof. Markus Rehm, where she is now investigating the mechanisms underlying cell fate decisions in response to death stimuli throughout the cell cycle at the single-cell level.

Cell cycle progression and transmitotic apoptosis resistance promote escape from extrinsic apoptosis

Extrinsic apoptosis relies on TNF-family receptor activation by immune cells or receptor-activating biologics. Here, we monitored cell cycle progression at minutes resolution to relate apoptosis kinetics and cell-to-cell heterogeneities in death decisions to cell cycle phases. Interestingly, we found that cells in S phase delay TRAIL receptor-induced death in favour for mitosis, thereby passing on an apoptosis-primed state to their offspring. This translates into two distinct fates, apoptosis execution post mitosis or cell survival from inefficient apoptosis. Transmitotic resistance is linked to Mcl-1 upregulation and increased accumulation at mitochondria from mid S phase onwards, which allows cells to pass through mitosis with activated caspase-8, and with cells escaping apoptosis after mitosis sustaining sublethal DNA damage. Antagonizing Mcl-1 suppresses cell cycle-dependent delays in apoptosis, prevents apoptosis-resistant progression through mitosis and averts unwanted survival from apoptosis induction. Cell cycle progression therefore modulates signal transduction during extrinsic apoptosis, with Mcl-1 governing decision making between death, proliferation and survival. Cell cycle progression thus is a crucial process from which cell-to-cell heterogeneities in fates and treatment outcomes emerge in isogenic cell populations during extrinsic apoptosis.

Priming Death Receptor Mediated Apoptosis with Arginine Starvation Sensitises Arginine Auxotrophic B-ALL to CAR-T

Background Chimeric antigen receptor (CAR)-T cell therapy has revolutionised the treatment of relapsed or refractory B-ALL in children and young adults with unprecedented response rates. However, primary resistance and relapse are unresolved challenges that limit long term benefit in a significant proportion of patients. Death receptor mediated extrinsic apoptosis is a key component of CAR-T cytotoxicity and impairment of this system, of which CAR-T derived TRAIL (tumour necrosis factor related apoptosis inducing ligand) is a key initiator, is a principal driver of primary resistance. Arginine deprivation with the therapeutic enzyme ADI-PEG20 (pegylated arginine deiminase) sensitises cancers deficient in the enzyme argininosuccinate synthase (ASS1) to the apoptosis initiating activity of TRAIL through tumour cell surface upregulation of death receptors DR4 and DR5. Whether this effect could potentiate the TRAIL-DR activity in CAR-T therapy has not been explored. Aim We tested the hypothesis that ADI-PEG20 treatment can sensitise susceptible B-ALL to anti-CD19 CAR-T through priming of death receptor mediated apoptosis signalling. Methods The effect of ADI-PEG20 on cell survival and death receptor expression in B-ALL cell lines and primary samples was analysed by flow cytometry. Second generation anti-CD19 CAR-T cells with a CD28 costimulatory domain were generated by retroviral transduction of activated peripheral blood mononuclear cells (PBMC) from healthy donors. For CAR-T co-culture experiments, B-ALL cell lines were pre-treated with ADI-PEG20 before washing and re-suspending in arginine replete media prior to CAR-T cell addition. Results To establish potential susceptibility of B-ALL to ADI-PEG20 we measured expression of ASS1, which inversely correlates with sensitivity to the drug, using combined in situ immunohistochemistry (n=6) and RT-qPCR (n=7). ASS1 deficiency was consistently seen in this series of primary samples suggesting the potential utility of ADI-PEG20 in B-ALL, with comparable expression levels to those seen in a cohort of primary AML samples proven to be sensitive to the drug (figure 1a). Next, to examine variation in ASS1 expression between genetically defined subtypes of B-ALL we re-analysed transcriptome data from a cohort of 215 patients treated on the ECOG E2993 trial, filtered into a network of 58 genes generated according to known or predicted interaction with ASS1. We found an enrichment of Philadelphia chromosome positive (Ph+) and Philadelphia-like (Ph-L) samples in the cluster characterised by lowest ASS1 expression along with high HIF1A expression, matching a recurrent pattern reported in other ADI-PEG20 sensitive tumours. This therefore predicts that among B-ALL subtypes, Ph+ and Ph-L are likely to be most sensitive to therapeutic arginine deprivation. We then functionally confirmed, using in vitro cell line (n=3) and in vivo patient derived xenograft models of B-ALL (n=2), that ASS1 deficiency is required for ADI-PEG20 sensitivity. Using the ASS1-low, Ph-L cell line MUTZ-5, we established that ADI-PEG20 induced apoptosis accompanies cell surface upregulation of both DR4 and DR5 expression. Upregulation of DR4 was observed to follow an upwards trend after treated cells were washed and re-suspended in arginine replete media, suggesting that transient arginine starvation can commit ASS1-low B-ALL to a state of apoptotic priming (figure 1b). With confirmed engagement of arginine starvation and death receptor upregulation we tested the synergy potential of ADI-PEG20 pre-treatment of MUTZ-5 followed by CAR-T, utilising calculated combination drug indices (CDIs). Across independent PBMC donors (n=3) we observed greater potency killing of CD19 + leukaemia cells in the combination treated co-cultures when compared to the single agent treated conditions, with CDIs consistently less than 1 confirming a synergistic effect (figure 1c). Conclusion Our study proposes a synergistic interaction between the arginine depleting enzyme ADI-PEG20 and anti-CD19 CAR-T for the treatment of ASS1 deficient B-ALL, whereby priming of death receptor signalling may underlie enhanced CAR-T cytotoxicity against CD19 + tumour cells. These data support an emerging framework for CAR-T optimisation based on targeting of the death receptor mediated extrinsic apoptosis pathway and can inform future refinements in the development of cellular immunotherapy. Figure 1 Figure 1. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Current Employment. Maher: Leucid Bio: Other: Chief Scientific Officer. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Co-Targeting Intrinsic and Extrinsic Apoptosis to Maximize Cell Death Induction in Venetoclax-Resistant AML Cells

Bcl-2 family protein-regulated intrinsic and IAP-family protein-regulated extrinsic pathways are two major apoptotic cell death mechanisms. Components of both pathways are regulated by the tumor suppressor p53. Although combinations of Bcl-2 inhibitor venetoclax (VEN) and a hypomethylating agent induce high response rates in AML, most patients ultimately relapse. In addition, pre-clinical and clinical studies have shown that TP53-mutant AML cells are less sensitive to VEN (Carter BZ, ASH 2019 and 2020; DiNardo CD, Blood 2020). We investigated if simultaneous inhibition of Bcl-2 and IAPs and activation of p53, via MDM2 inhibition could maximize apoptosis induction in AML cells with acquired resistance to VEN-based therapy or in those carrying TP53 mutations. We treated MV4-11 cells with acquired resistance to VEN (VEN-R) with the Bcl-2 inhibitor APG2575, IAP inhibitor APG1387, or MDM2 inhibitor APG115, and with their combinations. As expected, VEN-R cells were more resistant to APG2575 compared to control cells. APG1387 alone had limited activity in both VEN-R and in control cells and the combination of APG2575 and APG1387 enhanced cell killing (P < 0.05 compared to each single agent). APG115 was active in both VEN-R and control cells and its activity was only increased by APG2575 in the control cells, but minimally affected by either APG2575 or APG1387 in VEN-R cells. However, maximal apoptosis induction was observed in both VEN-R and control cells when all three compounds were combined (P < 0.05 compared to any of the double combinations or single agent treatments). We next treated NSG mice harboring PDX cells derived from an AML patient who relapsed on the VEN/decitabine therapy with APG2575 (50 mg/kg, p.o., daily), APG1387 (10 mg/kg, i.v., once/wk), APG115 (50 mg/kg, p.o., daily at wk 1 and 5), or combinations. At the end of a 5-wk treatment, significant reductions of human CD45 + cells were observed in all treatment groups. At 4 wks post treatment, decreased circulating leukemia cells were found in the triple and APG115+APG2575 combination groups. APG115 (139 d, p=0.009), APG1387 (130 d, p=0.004), or APG2575 (132 d, p=0.004) significantly extended mouse survival compared to controls (116 d). Among two drug combinations, APG115 plus APG1387 did not further prolong survival, APG2575 plus APG1387 (144 d, p<0.01) was more effective, and APG2575 plus APG115 (180 d, p<0.01) most effectively extended survival compared to each drug alone. Triple combination treated mice lived longest (185 d), which was significantly longer than APG115 plus APG1387 and APG2575 plus APG1387 but did not reach statistical significance compared to APG2575 plus APG115. Data showed that triple and APG115+APG2575 combinations were most effective, followed by APG2575+APG1387, then APG115+APG1387, APG2575, or APG115, and finally APG1387. Finally, we treated Molm13 cells lacking TP53 or carrying TP53 mutations (R248W/R213*, R248Q, R175H, R282W, Y220C) with the three agents and their combinations. All mutant cells were insensitive to single drugs. Enhanced activity was observed when any of two agents were combined and combined inhibition of Bcl-2, IAPs, and MDM2 most effectively induced cell death in TP53 knockout and all TP53 mutant cells (P < 0.05 for the triple combination compared to any of the double combinations or single agent treatments, and double combinations compared to their respective single agent treatments). Western blot analysis showed that decreased cIAP1, cIAP2, XIAP, or p21 was observed in single agent or combination-treated cells. Only in the triple combination group, cIAP1, cIAP2, and XIAP as well as MDM2 were largely diminished and p21 was marked decreased. In conclusion, our study demonstrates that co-targeting intrinsic and extrinsic apoptosis maximizes cell death induction in AML cells with acquired resistance to VEN or with TP53 deletion/mutations by antagonizing Bcl-2, eliminating cIAPs and XIAP, as well as MDM2 and p21, a finding that needs to be validated clinically. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Andreeff: Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Medicxi: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees.

Comparative Study of the Pharmacological Properties and Biological Effects of Polygonum aviculare L. herba Extract-Entrapped Liposomes versus Quercetin-Entrapped Liposomes on Doxorubicin-Induced Toxicity on HUVECs

This study aimed to evaluate the comparative biological effects of Polygonum aviculare L. herba (PAH) extract and quercetin-entrapped liposomes on doxorubicin (Doxo)-induced toxicity in HUVECs. HUVECs were treated with two formulations of liposomes loaded with PAH extract (L5 and L6) and two formulations of liposomes loaded with quercetin (L3 prepared with phosphatidylcholine and L4 prepared with phosphatidylserine). The results obtained with atomic force microscopy, zeta potential and entrapment liposome efficiency confirmed the interactions of the liposomes with PAH or free quercetin and a controlled release of flavonoids entrapped in all the liposomes. Doxo decreased the cell viability and induced oxidative stress, inflammation, DNA lesions and apoptosis in parallel with the activation of Nrf2 and NF-kB. Free quercetin, L3 and L4 inhibited the oxidative stress and inflammation and reduced apoptosis, particularly L3. Additionally, these compounds diminished the Nrf2 and NF-kB expressions and DNA lesions, principally L4. PAH extract, L5 and L6 exerted antioxidant and anti-inflammatory activities, reduced γH2AX formation and inhibited extrinsic apoptosis and transcription factors activation but to a lesser extent. The loading of quercetin in liposomes increased the cell viability and exerted better endothelial protection compared to free quercetin, especially L3. The liposomes with PAH extract had moderate efficiency, mainly due to the antioxidant and anti-inflammatory effects and the inhibition of extrinsic apoptosis.

Demethylzeylasteral (T-96) initiates extrinsic apoptosis against prostate cancer cells by inducing ROS-mediated ER stress and suppressing autophagic flux

Background Demethylzeylasteral (T-96) is a pharmacologically active triterpenoid monomer extracted from Tripterygium wilfordii Hook F (TWHF) that has been reported to exhibit anti-neoplastic effects against several types of cancer cells. However, the potential anti-tumour effects of T-96 against human Prostate cancer (CaP) cells and the possible underlying mechanisms have not been well studied. Results In the current study, T-96 exerted significant cytotoxicity to CaP cells in vitro and induced cell cycle arrest at S-phase in a dose-dependent manner. Mechanistically, T-96 promoted the initiation of autophagy but inhibited autophagic flux by inducing ROS-mediated endoplasmic reticulum (ER) stress which subsequently activated the extrinsic apoptosis pathway in CaP cells. These findings implied that T-96-induced ER stress activated the caspase-dependent apoptosis pathway to inhibit proliferation of CaP cells. Moreover, we observed that T-96 enhances the sensitivity of CaP cells to the chemotherapeutic drug, cisplatin. Conclusions Taken together, our data demonstrated that T-96 is a novel modulator of ER stress and autophagy, and has potential therapeutic applications against CaP in the clinic.

Multiple Autonomous Cell Death Suppression Strategies Ensure Cytomegalovirus Fitness

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.

Fangchinoline Inhibits Human Esophageal Cancer by Transactivating ATF4 to Trigger Both Noxa-Dependent Intrinsic and DR5-Dependent Extrinsic Apoptosis

Esophageal squamous cell carcinoma (ESCC) is a recalcitrant cancer. The Chinese herbal monomer fangchinoline (FCL) has been reported to have anti-tumor activity in several human cancer cell types. However, the therapeutic efficacy and underlying mechanism on ESCC remain to be elucidated. In the present study, for the first time, we demonstrated that FCL significantly suppressed the growth of ESCC both in vitro and in vivo. Mechanistic studies revealed that FCL-induced G1 phase cell-cycle arrest in ESCC which is dependent on p21 and p27. Moreover, we found that FCL coordinatively triggered Noxa-dependent intrinsic apoptosis and DR5-dependent extrinsic apoptosis by transactivating ATF4, which is a novel mechanism. Our findings elucidated the tumor-suppressive efficacy and mechanisms of FCL and demonstrated FCL is a potential anti-ESCC agent.