Mechanism Of Cell Killing Research Articles

Dissecting the Cell-Killing Mechanisms of Hydroxyurea Using Spot Assays.

Hydroxyurea is an inhibitor of ribonucleotide reductase and is commonly used in laboratories to induce replication stress or arrest cells in the S phase for cell cycle or checkpoint studies. However, hydroxyurea also causes side effects such as oxidative stress, particularly under chronic exposure conditions. This complicates the interpretation of the cell-killing mechanisms, particularly in a previously uncharacterized mutant, and thus hampers the analyses. Here, we describe a few easy and simple spot assays in fission yeast that allow dissecting the cell-killing mechanisms of hydroxyurea.

Hydrogen-Bonding-Regulated Morphology Control and the Impact on the Antibacterial Activity of Cationic π-Amphiphiles.

This manuscript describes the synthesis, self-assembly, and antibacterial properties of naphthalene-diimide (NDI)-derived cationic π-amphiphiles. Three such asymmetric NDI derivatives with a nonionic hydrophilic wedge and a guanidine group in the two opposite sides of the NDI chromophore were considered. They differ by a single functional group (hydrazide, amide, and ester for NDI-1, NDI-2, and NDI-3, respectively), located in the linker between the NDI and the hydrophilic wedge. For NDI-1, the H-bonding among the hydrazides regulated unilateral stacking and a preferential direction of curvature of the resulting supramolecular polymer, producing an unsymmetric polymersome with the guanidinium groups displayed at the outer surface. NDI-3, lacking any H-bonding group, exhibits π-stacking without any preferential orientation and generates spherical particles with a relatively poor display of the guanidium groups. In sharp contrast to NDI-1, NDI-2 exhibits an entangled one-dimensional (1D) fibrillar morphology, indicating the prominent role of the H-bonding motif of the amide group and flexibility of the linker. The antibacterial activity of these assemblies was probed against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). NDI-1 showed the most promising antibacterial activity with a minimum inhibitory concentration (MIC) of ∼7.8 μg/mL against S. aureus and moderate activity (MIC ∼ 125 μg/mL) against E. coli. In sharp contrast, NDI-3 did not show any significant activity against the bacteria, suggesting a strong impact of the H-bonding-regulated directional assembly. NDI-2, forming a fibrillar network, showed moderate activity against S. aureus and negligible activity against E. coli, highlighting a significant impact of the morphology. All of these three molecules were found to be compatible with mammalian cells from the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and hemolysis assay. The mechanistic investigation by membrane polarization assay, live/dead fluorescence assay, and microscopy studies confirmed the membrane disruption mechanism of cell killing for the lead candidate NDI-1.

Patient-reported outcomes (PROs) from the DREAMM-7 randomized phase 3 study comparing belantamab mafodotin, bortezomib, and dexamethasone (BVd) vs daratumumab, bortezomib, and dexamethasone (DVd) in patients with relapsed/refractory multiple myeloma (RRMM).

7543 Background: Belantamab mafodotin(belamaf), a first-in-class antibody-drug conjugate targeting B-cell maturation antigen, acts through a multimodal mechanism including direct cell killing and immune-mediated mechanisms. The global, phase 3, open-label, randomized, DREAMM-7 trial (NCT04246047) met its primary endpoint of demonstrating statistically significant progression-free survival benefit favoring BVd vs DVd in patients with RRMM who had received ≥1 prior line of therapy; here, we report PRO findings for BVd vs DVd. Methods: Patients were randomized (1:1) to receive BVd or DVd and completed electronic PRO measures at baseline and every 3 weeks (Q3W) during treatment. PRO measures included EORTC-QLQ-C30, EORTC-QLQ-MY20 disease symptoms (pain), PRO-CTCAE patient-reported tolerability, and OSDI vision-related functioning (Q3W up to the sixth dose of belamaf, then Q6W). Each domain was summarized using descriptive statistics. Results: Among 494 patients (BVd, n=243; DVd, n=251), adherence to PRO assessments was >90% for most study visits. There was no difference in EORTC QLQ-C30 global health status/quality-of-life assessments between the study arms over time. Similarly, role functioning, physical functioning, fatigue, and pain were stable (change from baseline in EORTC score was within 10 points) over time and consistent between arms. Most symptomatic adverse events evaluated by PRO-CTCAE were reported at no to low severity, frequency, and interference (PRO-CTCAE ratings ≤2) in both arms throughout the study. The severity and interference of blurred vision and frequency of watery eyes were reported at higher levels (PRO-CTCAE ratings ≥3) in the BVd arm. Among patients in the BVd arm with a clinically meaningful deterioration in vision-related functioning (a change from baseline of ≥12.5 points), EORTC-QLQ-C30 global health status, role functioning, and physical functioning were comparable to the DVd arm over time. Conclusions: Overallquality of life, role functioning, physical functioning, fatigue, and pain were comparable in patients treated with BVd vs DVd. In patients treated with BVd who reported a clinically meaningful deterioration in vision-related functioning, overall quality of life was consistent with the DVd arm.

Abstract PO5-27-10: Next generation antibody drug conjugates: Multi-payload conjugates targeting multiple mechanisms of cell killing

Abstract Background: ADCs have had tremendous impact on patient outcomes in breast cancer and are rapidly moving towards second line use. However, many patients fail to respond or relapse after treatment with ADC therapies due to tumor heterogeneity and resistance to ADC payloads. Method: CatenaBio has developed a novel Multi-Payload-Conjugate (MPC) system capable of attaching distinct payloads at different sites to the same antibody, enabling the production of single-molecule targeted combination therapies with defined DAR. Results: We screened combinations of different payloads targeting several different mechanisms attached to trastuzumab at different drug-antibody ratios to optimize tumor cell killing. These targeted combination ADCs demonstrated robust killing in both HER2 high and low tumor cell lines. Conclusion: Antibody Drug Conjugates have revolutionized the treatment of high HER2 positive breast cancer. More recently advances have been made in the design of ADCs to expand indications to include HER2 low as well as HER2 negative patients. These constructs offer the next step in ADC design and allow for the combination of multiple mechanisms of action in a single MPC that are highly effective across multiple breast cancer cell lines. These molecules offer the potential to circumvent tumor resistance pathways and deliver deeper and more durable responses. Disclosures: Marco Lobba, Devin Trinter, Maxwell Nguyen, Daniel Gutierrez, Samantha Brady, Chanez Symister, Andrew Lau, Richard Kendall, and Saurabh Johri are employed by Catena Biosciences. Matthew Francis is a co-founder and advisor to Catena Biosciences. Citation Format: Marco Lobba, Devin Trinter, Maxwell Nguyen, Daniel Gutierrez, Samantha Brady, Chanez Symister, Andrew Lau, Richard Kendall, Saurabh Johri, Matthew Francis. Next generation antibody drug conjugates: Multi-payload conjugates targeting multiple mechanisms of cell killing [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-10.

Potential of Ruthenium as a photosensitizer in radiation-activated Photodynamic Therapy (radioPDT)

In Photodynamic Therapy (PDT), light is used to activate photosensitizers (PS) to generate reactive oxygen species (ROS) as a cell-killing mechanism. However, PDT's application is limited to the surface and small size tumors because of the inefficiency of light in penetrating larger and deep-seated tumors. We have developed novel nanoparticles (NPs) that can be activated by radiation, a process known as the radiation-activated PDT (radioPDT). In our studies we developed a new variant of pegylated poly-lactic-co-glycolic (PEG-PLGA) encapsulated nanoscintillators (NSC) and ruthenium-based photosensitizer (Ru/rPDT) that exhibits minimal cell toxicity while having significant cell killing effect upon radiation activation. Our data suggest that radioPDT with Ru/rPDT can be a more effective strategy than previous protoporphyrin-based iterations in treating many deep-seated tumors.Introduction and Background: Toxicity and normal tissue damage are limiting factors for the application of radiotherapy (RT) where higher doses of radiation is required to cure deep-seated tumor such as prostate cancer. In Photodynamic Therapy (PDT), photosensitizers (PS) are activated by light to generate reactive oxygen species (ROS) to kill tumor cells. However, the main limitation of the current PDT is the limited tissue penetration of PS-activating light. Thus, PDT cannot be effectively used to treat deep-seated and larger size tumors. RT has higher tissue penetration depth, making the radiation-activated PDT (radioPDT) advantageous in treating deep seated tumors.We aim to develop novel radioPDT nanoparticles (NP) with more efficient anti-cancer therapeutic effect in vitro and in vivo to enhance targeted radiotherapy.Methods: LaF3:Ce3+ nanoscintillators (NSCs) were synthesized in a single step wet chemistry technique. The NSCs along with Ruthenium-based PS (Ru) with matching excitation/emission spectra were successfully encapsulated in PEG-PLGA. UV-Vis spectrometry and inductively coupled plasma mass spectrometry (ICP-MS) were used to confirm the presence of NSCs and Ru, and dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to determine the size of NP. Singlet oxygen generation was measured by Singlet Oxygen Sensor Green (SOSG) and direct singlet oxygen fluorescence. RadioPDT therapeutic potential was evaluated in PC3 prostate cancer cell under radiation by Alamar Blue cell viability assay.Results: UV-Vis spectrometry and ICP-MS determined the successful PEG-PLGA encapsulation of NSC and Ru. The TEM imaging confirmed the encapsulation NSC inside the nanocarrier PEG-PLGA. TEM and DLS measurements showed the size of radioNP ranged from 90nm to 150nm with polydispersity index of <0.3. Singlet oxygen generation by Ru/rPDT NPs was greater than PPIX based radioPDT (PPIX/rPDT) NPs. Furthermore, the direct measurement of singlet oxygen generation with fluorescence spectroscopy of Ru/rPDT showed preserved singlet oxygen yield and no evidence of quenching. The Ru/rPDT and PPIX/rPDT showed minimal PC3 cell cytotoxicity in dark conditions. The PC3 cell killing efficiency was comparable between Ru/rPDT and PPIX/rPDT either under light or radiation activation.Conclusion: Our in vitro studies show Ru/rPDT significantly increases anti-cancer therapeutic effect of RT, with minimal toxicity. Future studies will refine in vivo NP biodistribution, dosing, and RT dose/fractionation schemes.

Abstract 5797: Next generation antibody drug conjugates: Multi-payload conjugates targeting multiple mechanisms of cell killing

Abstract Introduction: ADCs have had tremendous impact on patient outcomes in breast cancer and are now second line therapy for stage IV HER2 positive metastatic breast cancer. However, many patients fail to respond or relapse after treatment with ADC therapies due to tumor heterogeneity and resistance to ADC payloads. We are developing next generation ADCs that deliver targeted combination chemotherapies with a single molecule. Method: CatenaBio has developed a novel system capable of attaching distinct payloads at different sites to the same antibody, enabling the production of highly stable single-molecule targeted combination therapies, Multi-Payload-Conjugates (MPCs), with a well-defined drug-antibody ratio (DAR). Results: We screened combinations of different payloads targeting several different mechanisms of cell division attached to trastuzumab at different DARs to optimize tumor cell killing. These targeted combination ADCs demonstrated robust killing in both HER2 high and low tumor cell lines. Additionally, these novel MPCs show potent inhibition of tumor growth in a HER2+ xenograft model of cancer. Conclusion: Antibody Drug Conjugates have revolutionized the treatment of high HER2 positive breast cancer. More recently advances have been made in the design of ADCs to expand indications to include HER2 low as well as HER2 negative patients. Multi-payload Conjugates® offer the next step in ADC design and allow for the combination of multiple mechanisms of action in a single MPC that are highly effective across multiple breast cancer cell lines and HER2 expression levels. These molecules offer the potential to circumvent tumor resistance pathways and deliver deeper and more durable responses. Citation Format: Richard L. Kendall, Marco Lobba, Saurabh Johri, Chanez Symister, Devin Trinter, Maxwell Nguyen, Daniel Gutierrez, Samantha Brady, Andrew Lau, Matthew B. Francis. Next generation antibody drug conjugates: Multi-payload conjugates targeting multiple mechanisms of cell killing [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 5797.

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Rhamnose modified antibodies show improved immune killing towards EGFR-positive solid tumor cells

Therapeutic monoclonal antibodies (mAbs) against the epidermal growth factor receptor (EGFR) have shown clinical efficacy in colorectal cancer and other solid cancers. Enhancing the effector functions of these anti-EGFR mAbs is believed to be a valuable approach to achieve improved efficacy in clinical setting. Here, we report the development of an effector function-enhanced antibody by rhamnose (Rha) functionalization. Cetuximab, a human/mouse chimeric anti-EGFR mAb, was selected and site-specifically conjugated with Rha haptens. The obtained cetuximab-Rha conjugate was shown to be able to selectively redirect amounts of endogenous anti-Rha antibodies onto EGFR-positive solid tumor cells and thereby provide more Fc domains to achieve enhancement of effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP). Particularly, CDC, one powerful cell killing mechanism which is inactive in cetuximab, was dramatically improved. This study demonstrates the potential of rhamnose-modified antibody for EGFR-positive solid tumor immunotherapy.

Abstract IA008: Targeting homologous recombination deficiency (HRD) using optimized ionizing radiation and drug combinations

Abstract Homologous recombination deficiency (HRD) is found not only in germline mutation carriers of HR genes but is also observed in sporadic cancers when both copies of the HR gene are inactivated. Recognition of HRD is facilitated by using genomic landscape alterations in addition to target gene sequencing. In addition, HR function can be established by measuring RAD51 foci in tumor specimens, particularly if they are irradiated ex-vivo to reveal the HR pathway function more clearly. Triple negative breast cancer (TNBC) is highly enriched for HRD constituting ~45% of primary tumors and over 50% of locally recurrent or metastatic cancers. In a clinical trial of recurrent TNBC, we used the combination of ionizing radiation (IR) with low-dose weekly cisplatin (cis) to study the clinical response according to HRD status. The combination of IR + cis has been shown in HRD cells to be more sensitizing than either agent alone. In the clinical study of 49 TNBC patients, there were imaging responses in 22/29 (75%) HRD patients, but only in 4/20 (20%) HR-proficient patients. We have followed this study by investigating the combination of the PARP-inhibitor with radiotherapy in an ongoing clinical trial as well as with detailed cell-based studies. HRD cells have elevated PARP activity at baseline, as observed by the extent of PARylated proteins, plus the lower baseline levels of NAD. When PARP-inhibitors are combined with radiotherapy, there is demonstrable supra-additive sensitization at 6Gy of IR and up to 100nM talazoparib (TZ). However, when the dose of TZ is above 1 µM, there is paradoxical recovery from the combination therapy, due to rescue of NAD depletion in HRD cells. The threshold for seeing this paradoxical recovery effect in HR-proficient cells was not reached. One of the mechanisms of cell killing in HRD cells is enhanced parthanatos, which higher doses of TZ will prevent, particularly in conjunction with IR. The implication of this work is that the optimal combination of radiotherapy with PARP-inhibitor may not be the maximum tolerated dose, but rather a tuned level of the combination to maximize radiosensitization without exceeding the threshold for PARP-inhibitor mediated protection of cell death. It is already observed that the maximum tolerated dose of PARP-inhibitor in the setting of curative doses of IR have to be modified by 3-10-fold. Further optimization of the combination dosing to achieve the maximal therapeutic ratio will be discussed including the development of biomarkers to achieve this goal. Additional radiation combinations with synthetic lethal targets of HRD will be discussed. Citation Format: Simon N. Powell, Niamh McDermott, Robert Delsite. Targeting homologous recombination deficiency (HRD) using optimized ionizing radiation and drug combinations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA008.

CD8+ T Cell Biology in Cytokine Storm Syndromes.

Familial forms of hemophagocytic lymphohistiocytosis (HLH) are caused by loss-of-function mutations in genes encoding perforin as well as those required for release of perforin-containing cytotoxic granule constituent. Perforin is expressed by subsets of CD8+ T cells and NK cells, representing lymphocytes that share mechanism of target cell killing yet display distinct modes of target cell recognition. Here, we highlight recent findings concerning the genetics of familial HLH that implicate CD8+ T cells in the pathogenesis of HLH and discuss mechanistic insights from animal models as well as patients that reveal how CD8+ T cells may contribute to or drive disease, at least in part through release of IFN-γ. Intriguingly, CD8+ T cells and NK cells may act differentially in severe hyperinflammatory diseases such as HLH. We also discuss how CD8+ T cells may promote or drive pathology in other cytokine release syndromes (CSS). Moreover, we review the molecular mechanisms underpinning CD8+ T cell-mediated lymphocyte cytotoxicity, key to the development of familial HLH. Together, recent insights to the pathophysiology of CSS in general and HLH in particular are providing promising new therapeutic targets.

Morphological Effects and In Vitro Biological Mechanisms of Radiation-Induced Cell Killing by Gold Nanomaterials.

Gold nanomaterials have been shown to augment radiation therapy both in vitro and in vivo. However, studies on these materials are mostly phenomenological due to nanoparticle heterogeneity and the complexity of biological systems. Even accurate quantification of the particle dose still results in bulk average biases; the effect on individual cells is not measured but rather the effect on the overall population. To perform quantitative nanobiology, we coated glass coverslips uniformly at varying densities with Au nanoparticle preparations with different morphologies (45 nm cages, 25 nm spheres, and 30 nm rods). Consequently, the effect of a specific number of particles per unit area in contact with breast cancer cells growing on the coated surfaces was ascertained. Gold nanocages showed the highest degree of radiosensitization on a per particle basis, followed by gold nanospheres and gold nanorods, respectively. All three materials showed little cytotoxic effect at 0 Gy, but clonogenic survival decreased proportionally with the radiation dose and particle coverage density. A similar trend was seen in vivo in the combined treatment antitumor response in 4T1 tumor-bearing animals. The presence of gold affected the type and quantity of reactive oxygen species generated, specifically superoxide and hydroxyl radicals, and the concentration of nanocages correlated with the development of more numerous double-stranded DNA breaks and increased protein oxidation as measured by carbonylation. This work demonstrates the dependence on morphology and concentration of radiation enhancement by gold nanomaterials and may lead to a novel method to differentiate intra- and extracellular functionalities of gold nanomedicine treatment strategies. It further provides insights that can guide the rational development of gold nanomaterial-based radiosensitizers for clinical use.

Single-Cell RNA Sequencing Reveals the Interplay between Circulating CD4 + T Cells, B Cells and Cancer-Associated Monocytes in Classic Hodgkin Lymphoma Treated with PD-1 Blockade

Classic Hodgkin lymphoma (cHL) is a largely MHC class I-negative tumor with recurrent 9p24.1/ PD-L1/ PD-L2 copy gains and the highest reported response rates to PD-1 blockade. In cHL, the efficacy of PD-1 blockade is closely associated with Hodgkin Reed-Sternberg (HRS) cell expression of MHC class II, highlighting the potential role of CD4 + T-cell effectors and additional non-MHC class I-mediated mechanisms of tumor cell killing. We utilized scRNA and scT-cell receptor (TCR) sequencing to characterize the peripheral immune response to PD-1 blockade and define non-CD8 + T-cell dependent mechanisms of immune evasion in cHL. Peripheral blood mononuclear cells were obtained from 20 patients with relapsed/refractory (R/R) cHL who were treated with PD-1 blockade on the CheckMate 205 clinical trial (at cycle 1 day 1 [C1D1] and cycle 4 day 1 [C4D1]), 11 patients with newly diagnosed (ND), previously untreated cHL and 13 healthy donors. We first analyzed circulating CD4 + T cells given their likely importance in the response to PD-1 blockade in cHL. We found that patients with R/R cHL had markedly lower baseline TCR diversity than healthy donors across the entire CD4 + naïve/central memory (CM) T-cell space. Additionally, patients with the most favorable responses to PD-1 blockade had significantly increased CD4 + naïve/CM TCR diversity at baseline (C1D1) and more abundant naïve/CM subsets on treatment (C4D1). These response-related differences likely reflect a continued capacity to mount CD4 + T-cell responses to neoantigens. We also identified several circulating CD4 + T-cell populations that were significantly more abundant in patients with cHL than in healthy donors, including follicular helper-like T cells, IFN-stimulated cytotoxic T cells and actively proliferating CTLA4 + LAG3 + T cells. Compared to healthy donors, all evaluated patients with cHL had significantly reduced numbers of classically defined circulating NK cells and an expanded population of IFN-stimulated NK cells with likely reduced cytotoxic potential. In addition to marked deregulation of the NK cell compartment, all patients with cHL had decreased numbers of circulating B cells at all stages of differentiation. We used the scRNA sequencing data to reconstruct individual B-cell receptor (BCR) sequences with the TRUST4 algorithm and found that the numbers of total and unique BCRs, as well as chao1 diversity, were significantly decreased in patients with ND cHL in comparison to healthy donors. Additionally, patients with the most favorable responses to PD-1 blockade had significantly higher baseline BCR diversity and circulating B-cell numbers. Altogether, these data underscore the likely importance of B cell-mediated immune responses in cHL, either directly or via Fc-binding of innate effectors. Finally, the most abundant circulating CD3 - population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumor-promoting cytokines/chemokines, as well as PD-L1 and SIRPa. These inflamed IL1β + monocytes were virtually absent from the blood of healthy donors. Using RNAscope, we also detected macrophages with similar features in the intact cHL tumor microenvironment, in the immediate proximity of malignant HRS cells. To assess the broader significance of the inflamed IL1β + monocytes, we interrogated a single-cell compendium of monocytes and macrophages from multiple solid tumors and identified a population of monocytes/ macrophages with a similar transcriptional signature. These results suggest that similarly programmed inflammatory monocytes/macrophages are detectable in multiple cancers. We next assessed the clinical significance of circulating inflamed IL1β + monocytes in patients with R/R cHL who were treated with PD-1 blockade. Patients who did not respond to PD-1 blockade expressed significantly higher levels of the inflamed IL1β + monocyte transcriptional signature which led to the development of a predictive assay. We then identified a comparable circulating monocyte population with the same transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor, renal cell carcinoma, which underscored the broad significance of these findings. Taken together, our findings highlight the likely complementary role of CD4 + T cells, B cells and cancer-associated monocytes in the response to PD-1 blockade in cHL.

Population Pharmacokinetics (PopPK) and Exposure-Response (E-R) Analyses for Belantamab Mafodotin Monotherapy in Patients with Relapsed/Refractory Multiple Myeloma (RRMM) Enrolled in DREAMM-2 and DREAMM-3

Background: Belantamab mafodotin (belamaf) is a B-cell maturation antigen (BCMA)-targeting antibody-drug conjugate (ADC) with a humanized afucosylated anti-BCMA monoclonal antibody (mAb) conjugated to a cytotoxic payload (microtubule-disrupting agent monomethyl auristatin F [MMAF]) by a protease-resistant maleimidocaproyl (mc) linker. This ADC binds to BCMA and induces cell death by direct cell killing and immune-mediated mechanisms. The DREAMM-2 trial (NCT03525678) showed rapid, deep, and durable responses to single-agent belamaf in patients with RRMM. The Phase 3 DREAMM-3 trial (NCT04162210) evaluated single-agent belamaf vs. pomalidomide plus low-dose dexamethasone in patients with RRMM at second relapse or later (Weisel et al., J Clin Oncol 41, 2023 [suppl 16; abstr 8007]); DREAMM-3 did not meet its primary endpoint on superiority for progression free survival (PFS). The present analysis evaluated the PopPK of the ADC and cytotoxin cysteine-mcMMAF (cys-mcMMAF) as well as the E-R relationships for key efficacy and safety endpoints to contribute to further understanding of belamaf. Methods: Patients with RRMM were administered intravenous (IV) doses of belamaf 2.5 or 3.4 mg/kg Q3W in DREAMM-2 (n=218) and 2.5 mg/kg Q3W in DREAMM-3 (n=217). Previously built PopPK models of ADC and cys-mcMMAF were updated and used to predict individual Cycle 1 exposure measures for the E-R analyses performed on the DREAMM-3 belamaf arm only. Exposure-efficacy analyses evaluated the probability of response (PoR), PFS, and time to response (TTR). Exposure-safety analyses evaluated the probability of occurrence of adverse events of special interest such as changes in best corrected visual acuity (BCVA), grade ≥2 or ≥3 corneal events, grade ≥3 thrombocytopenia and infusion-related reactions (IRR). Logistic regression models were used to assess event probabilities, while Cox proportional-hazard models were used to assess time to event endpoints. Stepwise covariate analyses were performed. Results: The PopPK data were described well by two-compartment models with linear elimination. The ADC model included time-varying clearance and the cys-mcMMAF model included decreasing MMAF:mAb ratio after each dose. ADC clearance was reduced on average by 30% over the first 5 months on treatment. The PopPK of ADC and cys-mcMMAF were both influenced by disease-related factors (baseline concentrations of soluble BCMA [sBCMA], immunoglobulin G, and albumin), as well as body weight and to a lower extent body mass index. Mild to severe renal impairment, mild hepatic impairment, age, race (Black or Asian), or prior therapies were not found to impact the PopPK of belamaf. Cycle 1 ADC average concentrations (C avg) were inversely correlated with disease burden factors and highly correlated (r=0.97) with ADC trough concentration (C tau). In DREAMM-3, PoR and PFS were higher and longer with higher ADC exposure when evaluated independently of other covariates. In the final model, PoR and PFS were respectively lower and shorter with higher baseline sBCMA or prior anti-CD38 mAb treatment; PoR was also related to presence of extra medullary disease. TTR was not related to ADC exposure or other factors. Safety endpoints were associated with exposure. Higher ADC C avg was associated with higher probability of developing grade ≥2 or ≥3 corneal events and inversely associated with time to their onset. ADC C avg was similarly associated with changes in BCVA but with a lower intercept for BCVA-related endpoints. Baseline sBCMA was inversely associated with probability of grade ≥2 corneal events. Lower baseline platelet count (but not ADC exposure) was associated with increased probability of grade ≥3 thrombocytopenia. ADC C avg was inversely associated with probability of IRR. In addition, the observed incidence of post-treatment anti-belamaf antibodies was low (&amp;lt;1%) with no observed impact on PK, efficacy, or safety. Conclusion: PopPK and E-R analyses for DREAMM-3 demonstrated the impact of disease factors and patient characteristics on PK, efficacy, and safety endpoints following single-agent belamaf, consistent with prior results for later-line populations.

Evaluating the cytotoxicity mechanism of the cell-penetrating peptide TP10 on Jurkat cells

TP10, a classic cell-penetrating peptide, shows a high degree of similarity to AMPs in structure. Although TP10 has been widely used in drug delivery, the mechanism underlying its cytotoxicity is yet to be elucidated. Herein, we explored the cell-killing mechanism of TP10 against human leukemia Jurkat cells. TP10 induced necrosis in Jurkat cells via rapid disruption of cell membranes, particularly at high concentrations. Although mitochondria in Jurkat cells were damaged by TP10, mitochondria-mediated apoptosis did not occur, possibly due to intracellular ATP depletion. Necroptosis in TP10-treated Jurkat cells became an alternative route of apoptosis. Our results demonstrate that necrosis and necroptosis rather than apoptosis are involved in the cell-killing mechanism of TP10, which contributes to the understanding of its toxicity.

Enterocin LD3 from Enterococcus hirae LD3 Inhibits the Growth of Salmonella entericasubsp. enterica serovar Typhimurium ATCC 13311 in Fruit Juice.

In order to prevent the growth of pathogens in food, bacteriocins produced by various probiotic lactic acid bacteria have been recognized as potential substitutes of chemical preservatives. In this study, enterocin LD3 was purified from the cell-free supernatant of a food isolate, Enterococcus hirae LD3 using multistep chromatography. In the fruit juice, lethal concentration (LC50) of enterocin LD3 was found to be 260µg/mL against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The cells treated with enterocin LD3 were red colour indicating dead cells after propidium iodide staining, while untreated cells were found blue after staining with 4', 6-diamidino-2-phenylindole. The mechanism of cell killing was analyzed using infrared spectrum of cells treated with enterocin LD3 which was found altered in the range of 1,094.30 and 1,451.82cm-1 corresponding to nucleic acids and phospholipids, respectively. The morphology of target cells were severely ruptured and lysed as observed under electron microscopy. Thus, the present study suggested that enterocin LD3 showed bactericidal activity against Salm. enterica subsp. enterica serovar Typhimurium ATCC 13311 and may be applied as a bio-preservative for the safety of fruit juices.

The LiaFSR-LiaX System Mediates Resistance of Enterococcus faecium to Peptide Antibiotics and to Aureocin A53- and Enterocin L50-Like Bacteriocins.

Multidrug-resistant Enterococcus faecium strains are currently a leading cause of difficult-to-treat nosocomial infections. The emerging resistance of enterococci to last-resort antibiotics, such as daptomycin, prompts a search for alternative antimicrobials. Aureocin A53- and enterocin L50-like bacteriocins are potent antimicrobial agents that form daptomycin-like cationic complexes and have a similar cell envelope-targeting mechanism of action, suggesting their potential as next-generation antibiotics. However, to ensure their safe use, the mechanisms of resistance to these bacteriocins and cross-resistance to antibiotics need to be well understood. Here, we investigated the genetic basis of E. faecium's resistance to aureocin A53- and enterocin L50-like bacteriocins and compared it with that to antibiotics. First, we selected spontaneous mutants resistant to the bacteriocin BHT-B and identified adaptive mutations in the liaFSR-liaX genes encoding the LiaFSR stress response regulatory system and the daptomycin-sensing protein LiaX, respectively. We then demonstrated that a gain-of-function mutation in liaR increases the expression of liaFSR, liaXYZ, cell wall remodeling-associated genes, and hypothetical genes involved in protection against various antimicrobials. Finally, we showed that adaptive mutations or overexpression of liaSR or liaR alone results in cross-resistance to other aureocin A53- and enterocin L50-like bacteriocins, as well as antibiotics targeting specific components of the cell envelope (daptomycin, ramoplanin, gramicidin) or ribosomes (kanamycin and gentamicin). Based on the obtained results, we concluded that activation of the LiaFSR-mediated stress response confers resistance to peptide antibiotics and bacteriocins via a cascade of reactions, eventually leading to cell envelope remodeling. IMPORTANCE Pathogenic enterococci carry virulence factors and a considerable resistome, which makes them one of the most serious and steadily increasing causes of hospital epidemiological risks. Accordingly, Enterococcus faecium is classified into a top-priority ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of six highly virulent and multidrug-resistant (MDR) bacterial pathogens for which novel antimicrobial agents need to be developed urgently. Alternative measures, such as the use of bacteriocins, separately or in combination with other antimicrobial agents (e.g., antibiotics), could be a potential solution, especially since several international health agencies recommend and support the development of such interventions. Nevertheless, in order to exploit their efficacy, more basic research on the mechanisms of cell killing and the development of resistance to bacteriocins is needed. The present study fills some of the knowledge gaps regarding the genetic basis of the development of resistance to potent antienterococcal bacteriocins, pointing out the common and divergent features regarding the cross-resistance to antibiotics.

The role of the host-Neutrophil biology.

Neutrophilic polymorphonuclear leukocytes (neutrophils) are myeloid cells packed with lysosomal granules (hence also called granulocytes) that contain a formidable antimicrobial arsenal. They are terminally differentiated cells that play a critical role in acute and chronic inflammation, as well as in the resolution of inflammation and wound healing. Neutrophils express a dense array of surface receptors for multiple ligands, ranging from integrins to support their egress from bone marrow into the circulation and from the circulation into tissues, to cytokine/chemokine receptors that drive their navigation to the site of infection or tissue damage and also prime them for a second stimulus, to pattern recognition receptors and immunoglobulin receptors to facilitate the destruction and removal of infective agents or debridement of damaged tissues. When afferent neutrophil signals are proportionate and coordinated they will phagocytose opsonized and unopsonized bacteria, activating the nicotinamide adenine dinucleotide phosphate oxidase (respiratory burst) to generate reactive oxygen species, which augment the proteolytic destruction of microbes secured within the phagosome. A highly orchestrated process of apoptosis follows, forming membrane-bound substructures that are removed by macrophages. Neutrophils are capable of various other forms of programmed cell death, such as NETosis and pyroptotic cell death, as well as nonprogrammed cell death by necrosis. In recent years, research has revealed that neutrophils are capable of far more subtle cell-cell interactions than previously thought possible. This includes synthesis of various inflammatory mediators and also myeloid cell training within bone marrow, where epigenetic and metabolic signals associated with returning neutrophils that undergo reverse egress from tissues into the vasculature and back to bone marrow program a hyperreactive subset of neutrophils during myelopoiesis that are capable of hypersensitive reactions to microbial aggressors. These characteristics are evident in various neutrophil subsets/subpopulations, creating broad heterogeneity in the behavior and biological repertoire of these seemingly schizophrenic immune cells. Moreover, neutrophils are critical effector cells of adaptive and innate immunity, binding to opsonized bacteria and destroying them by extracellular and intracellular methods. The former creates substantial collateral host tissue damage, as they are less specific than T-cytotoxic cell-killing mechanisms, and in conditions such as peri-implantitis, where plasma cells and neutrophils dominate the immune infiltrate, bone and tissue destruction are rapid and appear relentless. Finally, the role of neutrophils as conduits for periodontal-systemic disease connections and for oxidative damage to act as a causal link between the two has only recently been realized. In this chapter, we attempt to expand on these issues, emphasizing the contributions of European scientists throughout a detailed appraisal of the benefits and side effects of neutrophilic inflammation and immune function.

An in-vivo study of the combined therapeutic effects of pulsed non-thermal focused ultrasound and radiation for prostate cancer

Purpose The purpose of this study was to investigate the in vivo combined effects of pulsed focused ultrasound (pFUS) and radiation (RT) for prostate cancer treatment. Materials and Methods An animal prostate tumor model was developed by implanting human LNCaP tumor cells in the prostates of nude mice. Tumor-bearing mice were treated with pFUS, RT or both (pFUS + RT) and compared with a control group. Non-thermal pFUS treatment was delivered by keeping the body temperature below 42 °C as measured real-time by MR thermometry and using a pFUS protocol (1 MHz, 25 W focused ultrasound; 1 Hz pulse rate with a 10% duty cycle for 60 sec for each sonication). Each tumor was covered entirely using 4–8 sonication spots. RT treatment with a dose of 2 Gy was delivered using an external beam (6 MV photon energy with dose rate 300MU/min). Following the treatment, mice were scanned weekly with MRI for tumor volume measurement. Results The results showed that the tumor volume in the control group increased exponentially to 142 ± 6%, 205 ± 12%, 286 ± 22% and 410 ± 33% at 1, 2, 3 and 4 weeks after treatment, respectively. In contrast, the pFUS group was 29% (p < 0.05), 24% (p < 0.05), 8% and 9% smaller, the RT group was 7%, 10%, 12% and 18% smaller, and the pFUS + RT group was 32%, 39%, 41% and 44% (all with p < 0.05) smaller than the control group at 1, 2, 3, and 4 weeks post treatment, respectively. Tumors treated by pFUS showed an early response (i.e. the first 2 weeks), while the RT group showed a late response. The combined pFUS + RT treatment showed consistent response throughout the post-treatment weeks. Conclusions These results suggest that RT combined with non-thermal pFUS can significantly delay the tumor growth. The mechanism of tumor cell killing between pFUS and RT may be different. Pulsed FUS shows early tumor growth delay, while RT contributes to the late effect on tumor growth delay. The addition of pFUS to RT significantly enhanced the therapeutic effect for prostate cancer treatment.

Abstract A43: Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML

Abstract Background: RAS/RAF/ERK pathway activation is associated with primary or secondary resistance to venetoclax in AML. Oncogenic RAS activates mitochondrial fission through ERK1/2 mediated phosphorylation of the mitochondrial fission GTPase Drp1. Although the deletion of Drp1 inhibits KRAS driven pancreatic cancer, the role of RAS-driven mitochondrial dynamics in AML is unclear. Inhibiting ERK1/2 may, therefore, shed light on AML cells' dependence on RAS driven mitochondrial dynamics and metabolism. Objectives: We assessed the anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and further investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., 2018), sensitized intrinsically resistant and NRAS-mutant OCI-AML3 cells to venetoclax with combination index (CI) of 0.008. Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax (75% apoptosis versus 22% by venetoclax, CI=0.6). Bulk and CD34+CD38- leukemia initiating cells (LICs) were also eliminated in primary AML samples by the combination (CI:0.03-0.23). Colony formation assay and CyTOF using NRAS-mutant PDX showed impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to ERK1/2 inhibition and combination treatment, suggesting impact on stemness. ERK1/2 inhibition using ASTX029 (Heightman et al., 2021) alone and in combination with venetoclax reduced leukemia burden in-vivo and extended survival (p&amp;lt;0.05). Mitochondrial ROS mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML. ERK1/2 inhibition decreased pDrp1-Ser616 levels along with an increase in mitochondrial length (p&amp;lt;0.001) and mitochondrial ROS (p&amp;lt;0.01), suggesting impaired mitochondrial fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Overexpression of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells decreased apoptosis and reversed synergy between ERK1/2 and Bcl-2 inhibition as compared to wild type and phospho-null (Ser616Ala) Drp1 (p&amp;lt;0.001). Of note, overexpression of Drp1-phospho-mimetic led to decreased mitochondrial length, suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, significantly decreased maximal oxygen consumption rate, ATP production and ROS production. Moreover, combination treatment failed to significantly induce ROS production in cells expressing phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Conclusion: Inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. In summary, these data support combining ERK1/2 and Bcl-2 inhibitors in the treatment of AML and warrant further clinical investigation. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Anna Zal, Christopher Hindley, Kim-Hien Dao, Martin Sims, Tomasz Zal, Vivian Ruvolo, Michael Andreeff, Gautam Borthakur. Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A43.

Abstract 1371: Tumor treating fields exposure causes an imbalance of reactive oxygen homeostasis likely through the cytosolic function of the fanconi′s anemia genes

Abstract The use of Tumor Treating Fields (TTFields) has been shown to significantly benefit patients with non-resectable locally advanced or metastatic glioblastoma and malignant pleural mesothelioma. Although the disruption of mitosis that was initially attributed as the mechanism of tumor cell killing, in subsequent studies, it has been shown that TTFields inhibits DNA damage repair by downregulating DNA repair genes and proteins, including those associated with Fanconi's Anemia (FA). As a result, radiation-induced DNA damage repair is reduced and DNA replication fork protection is impaired, leading to replication stress and replication fork collapse. As the FA genes play a critical role in multiple repair pathways, TTFields is likely to increase susceptibility to other chemo agents besides radiation that cause DNA damage which is repaired by FA-dependent DNA repair and replication fork maintenance pathways in nuclei. In addition to their nuclear role, the FA proteins also regulate mitophagy in the cytosol. Using a panel of non-small cell lung cancer cell lines, it was found that TTFields exposure disrupted the clearance of damaged mitochondria despite the presence of a well-defined membrane markers for autophagosomal destruction of damaged mitochondria, that being PINK1 and parkin. Analysis of electron micrographs revealed disrupted mitochondrial cristae, while radical oxygen metabolism studies identified an imbalance leading to the production of reactive oxygen species (ROS) and ultimately, cell death. The downregulation of key mitochondrial genes including DAPIT, OSCP1, ATP5S, ATP5B and COXIV that are related to electron transport and mitochondrial integrity was confirmed by gene expression analysis. The small molecule dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, changes the metabolic state of cancer cells from cytoplasmic glycolysis to mitochondrial glucose oxidation, resulting in a decrease in mitochondrial membrane potential and an increase in ROS production in cancer cells. A combination of DCA with TTFields is therefore being investigated as a means of exploiting mitochondrial dysfunction caused by TTFields. The inhibition of mitophagy suggests a new mechanism of action for TTFields exposure tied to the downregulation of the FA pathway genes, the result being both nuclear and cytosolic perturbations in cell homeostasis. This study offers an evidence-based rationale for combining TTFields with various chemotherapy agents that may cause DNA damage, induce replication stress, and expose cells to radical oxygen byproducts of disrupting electron transport chains and the disruption of mitochondrial integrity. Citation Format: Narasimha Kumar Karanam, Michael Story. Tumor treating fields exposure causes an imbalance of reactive oxygen homeostasis likely through the cytosolic function of the fanconi′s anemia genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1371.