Antimitotic Research Articles

THE EFFECTIVENESS OF COLCHICINE AND ORYZALIN ON POLYPLOIDY INDUCTION IN TEAK (Tectona grandis Linn. f.) IN VITRO

Indonesian government has launched a community plantation forests program that would be beneficial for remedying shortage in domestic demand for teak wood every year. This program needs to be supported by the availability of quality seeds resources and quality seedlings utilizing polyploid teak (Tectona grandis). Our study aimed to examine the effectiveness of colchicine and oryzalin to modify diploid into polyploid T. grandis based on growth response, morphological, anatomical and cytological alteration, as well as the acclimatization ability of polyploid plantlets. The materials used were aseptic lateral shoots consisting of nodal segments which were immersed in antimitotic agents at the concentration of 0, 15 and 30 μM for 5 days, then cultured on regeneration medium until the 8 weeks and followed by acclimatization. The results showed that colchicine at the concentration of 30 μM was the most effective to induce polyploidy of plantlets in the parameter of high growth rate, the length of internodes, the number of leaf plantlets and increase the leaf surface area and the significant chlorophyll index content compared to the control. Anatomical analysis of polyploid was characterized by increasing the leaf thickness, stomata size, decreased stomatal density and increased chloroplast content in guard cells. Based on cytological examination on polyploidy plantlet, there was an increased in the number of chromosomes in the cell nucleus. The acclimatization of polyploid successfully induced rooting and a 100% survival rate of grown plantlets. Polyploid seedlings were able to grow and well adapted to the new environment of acclimatization

Computational Studies for Development of Triazole‐Pyrimidines as Inhibitor of α‐Tubulin Receptor

AbstractMicrotubules are among the critical targets for chemotherapeutic agents to fight against cancer because of their crucial role in mitosis. Microtubule inhibitors block cell cycle progression and result in apoptosis, they can frequently disrupt microtubule/tubulin dynamics by binding to the α‐tubulin receptor. To this end, we have developed compounds against α‐tubulin receptor using several tools of computational chemistry: 3D‐QSAR, Molecular Docking, Molecular Dynamics Simulation, and ADMET prediction. The significant findings incorporated two reliable models: CoMFA (Q2=0.75; R2=0.89) and CoMSIA (Q2=0.57; R2=0.78). Molecular Docking was generated and validated using Molecular Dynamics simulation to investigate the binding mode between the most active compound and the α‐tubulin receptor. The results of the MD simulation demonstrated stability over a 100 ns period. Four compounds (C1, C2, C3, and C4) were developed based on the contour maps, and their activities were predicted. Additionally, Molecular Docking was executed to elucidate the interaction and affinity between the novel compounds and the α‐tubulin receptor. C1 exhibited high affinity and essential interactions and was subjected to MD simulation studies. The results of the MD simulation revealed that the modifications made to enhance the anticancer activity did not impact stability. Finally, ADMET analysis was utilized to examine drug‐likeness.

Abstract A045: EZH2 inhibition re-sensitizes drug resistant triple-negative breast cancer PDX models to Eribulin

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer predominantly affecting younger women. The mainstay of treatment is chemotherapy, to which most patients eventually stop responding. After demonstrating improved overall survival in the metastatic setting, eribulin, a microtubule inhibitor, was approved for the treatment of late-stage, heavily pretreated breast cancer. While frequently used in the clinic, most patients either do not respond initially or they stop responding to eribulin. Considering that there is a lack of oncology drugs including targeted therapies to treat TNBCs, patients are therefore left with few therapeutic options after eribulin. There is a paucity of data on mechanisms of acquired resistance to eribulin, and few potential uncovered targetable alterations. The goal of our study is to determine mechanisms of acquired resistance to Eribulin using isogenic acquired-resistant PDX models and to attempt to reverse this resistance with novel combinations. Methods: PDX models are grown as mammary fat pad engraftments of patient tumours in immuno-compromised (NGS) mice. To determine drug response, we treat tumour-bearing mice with weekly eribulin, measure the tumor volume once per week, then classify response based on mRECIST 1.1 criteria. Acquired resistance was generated in vivo in TNBC PDXs via the continuous or interrupted treatment of Eribulin in sensitive models that regressed on Eribulin. RNA-Seq analysis was performed on isogenic pairs of Eribulin sensitive and resistant PDX tumors and analyzed for differential gene expression and pathway enrichments. Drug combinations targeting candidate regulators of enriched pathways across our models were assessed in vivo in resistant PDXs. Drug trials were performed with n=5-7 mice per treatment arm until endpoint (tumour volume reaches 2000mm^3 or BW loss of 20%). Statistical analysis included t-tests for tumour volume and Wilcoxon test for survival. Results: 6 isogenic pairs of Eribulin acquired resistant TNBC PDX models were generated in vivo and molecular analysis was performed by comparing transcriptomic profiles of sensitive and resistant tumours. Pathway analysis revealed that, across 5 of 6 acquired resistant models, differentially expressed genes were significantly enriched for targets of SUZ12 (BH p-value<0.01). As SUZ12 makes up a core component of the PRC2 complex, we attempted to target EZH2, the catalytic component of PRC2, in vivo. Combining Tazemetostat, an FDA-approved EZH2 inhibitor, with Eribulin in one PDX model, resulted in tumor regression and improved overall survival (p<0.005) in one model. We will be testing this combination in more models, and performing differential chromatin accessibility studies to identify candidate driver genes in these regions. Conclusion: Our results suggest that combining an approved EZH2 inhibitor, Tazemetostat, with Eribulin can re-sensitize acquired-resistant TNBC PDXs to Eribulin. This approach could have significant clinical benefit in late stage hard to treat TNBCs. Citation Format: Kathryn Bozek, Cedric Darini, Cathy Lan, Marguerite Buchanan, Catherine Chabot, Josiane Lafleur, Juliet Guay, Eva Filosa, Adriana Aguilar-Mahecha, Mark Basik. EZH2 inhibition re-sensitizes drug resistant triple-negative breast cancer PDX models to Eribulin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A045.

Design, Synthesis, in Silico and in Vitro Evaluation of New Combretastatin A-4 Analogs as Antimitotic Antitumor Agents.

Combretastatin A-4 (CA-4) binds β-tubulin at the colchicine-binding site preventing tubulin from polymerizing into microtubules. CA-4 and cis combretastatin analogs isomerize to the trans form resulting in decreased cytotoxicity and anti-tubulin activity. However, the excellent anti-cancer potential and relatively simple molecular structure of CA-4, provide an encouraging starting point for the development of new, more stable and more potent anti-tubulin compounds. To synthesize a new series of 4-(3,4,5-trimethoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives (compounds 10-12) as analogs of CA-4, with different alkyl, and aryl side chain substituents at the triazole moiety (B-ring), resulting in permanent cis form. The newly synthesized CA-4 analogs were characterized by FT-IR, 1HNMR, 13CNMR, and HR-MS(ESI) techniques. Molecular docking studies including docking score (G) ADMET, DFT, and mlocular similarities were performed. The anti-proliferative activity of the new compounds against three human cancer cell lines was evaluated using the MTT assay, and their ability to inhibit tubulin polymerization, and consequently their effects on cell cycle progression and induction of apoptois were assessed. Molecular docking studies showed that compounds 11b and 11d exhibited the highest docking scores (-13.30 and -14.01 Kcal/mol, respectively) into colchicine-binding site, scores very close to the reference drug colchicine (-13.50 Kcal/mol), and that hydrogen bonding and hydrophobic interaction are essential for binding. The most active cytotoxic compound 11b had potent IC50 values ranging from 3.83 to 10.67 µM against the three human cancer cell lines, while exhibiting low cytotoxicity against non-cancer human WI-38, suggesting that compound 11b targets rapidly growing cancer cells. Moreover, compound 11b exhibited potent anti-tubulin activity which was comparable to CA-4. Targeting microtubules caused cell cycle arrest at the G2/M phase resulting in induction of apoptosis. These findings indicate that compound 11b is a promising β-tubulin-binding compound with antimitotic action that has the potential to treat cancer.

Abstract C024: Trial in progress: Phase 1 study of BAL0891 as monotherapy and in combination with chemotherapy in patients with advanced solid tumors

Abstract Background: BAL0891 is a first-in-class small-molecule mitotic checkpoint inhibitor (MCI) that targets threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) to disrupt spindle assembly checkpoint (SAC) regulation and induce tumor cell death. It has a more potent anti-tumor activity as compared to TTK-specific inhibitors due to its combined prolonged effect on TTK and transient effect on PLK1. In preclinical studies, BAL0891 demonstrated potent anti-tumor activity as a monotherapy and in combination with paclitaxel and carboplatin. In the first-in-human microdose study of TTK-CS-001 in healthy subjects, key BAL0891 pharmacokinetic (PK) parameters were investigated (i.e., clearance, half-life, exposure), and no safety signals were detected. Trial design: This clinical trial is a multiple-cohort, open-label phase 1 study with 3 dose-escalation substudies, investigating intravenous (IV) BAL0891 as monotherapy, and in combination with carboplatin or paclitaxel, in patients with advanced solid tumors. Primary objectives: 1) To evaluate safety/tolerability 2) To estimate the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D) of BAL0891 monotherapy and in combination with carboplatin/paclitaxel. Eligibility: Patients ages ≥18 years with advanced/metastatic solid tumors that are refractory to, or intolerant of existing therapy may participate in this study. Patients enrolled in dose level ≥4 of Substudy 1 and all patients in Substudies 2 and 3 must have measurable disease. Substudy 1: Monotherapy BAL0891: IV doses of BAL0891 on Days 1 (D1) and 8 (D8) every 3 weeks (Q3W). A Bayesian logistic regression model (BLRM) will guide dose escalations, with 8 nominal DLs of 5/10/20/40/80/160/320/480 mg. Substudy 2: Phase 1 BAL0891 D1, D8 + carboplatin AUC5-6 D1 Q3W. Substudy 3: Phase 1 BAL0891 D1, D15 + paclitaxel 70-80 mg/m2 D1, D8, D15 Q4W. Substudies 2 and 3 may begin once the maximum tolerated dose (MTD) of BAL0891 monotherapy has been established or at the first signs of expected target toxicity and/or efficacy with monotherapy. Enrollment began in February 2023 and is ongoing in the United States and South Korea. Clinical trial information; NCT05768932 Citation Format: Shivaani Kummar, Minal Barve, Eric Feldman, Seunghyun Ma, Jaime Merchan, Seock-Ah Im, Sun Young Rha. Trial in progress: Phase 1 study of BAL0891 as monotherapy and in combination with chemotherapy in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C024.

Mitotic kinase inhibitors as Therapeutic Interventions for Prostate Cancer: Evidence from In vitro Studies.

Prostate cancer is one of the devastating diseases characterized by genetic changes leading to uncontrolled growth and metastasis of the cells of the prostate gland and affects men worldwide. Conventional hormonal and chemotherapeutic agents are effective in mitigating the disease if diagnosed at an early stage. All dividing eukaryotic cells require mitotic progression for the maintenance of genomic integrity in progeny populations. The protein kinases, upon activation and de-activation in an ordered fashion, lead to spatial and temporal regulation of the cell division process. The entry into mitosis along with the progression into sub-phases of mitosis is ensured due to the activity of mitotic kinases. These kinases include Polo-Like-Kinase 1 (PLK1), Aurora kinases, and Cyclin-Dependent-Kinase 1 (CDK1), among others. The mitotic kinases, among others, are usually overexpressed in many cancers and can be targeted using small molecule inhibitors to reduce the effects of these regulators on mechanisms, such as regulation of genomic integrity and mitotic fidelity. In this review, we attempted to discuss the appropriate functions of mitotic kinases revealed through cell culture studies and the impact of their respective inhibitors derived in pre-clinical studies. The review is designed to elucidate the growing field of small molecule inhibitors and their functional screening or mode of action at the cellular and molecular level in the context of Prostate Cancer. Therefore, studies performed specifically on cells of Prostatic-origin are narrated in this review, culminating in a comprehensive view of the specific field of mitotic kinases that can be targeted for therapy of Prostate cancer.

Abstract B146: Design and preclinical evaluation of CPL976-MMAE - novel, potent AXL-PD-L1 bispecific antibody conjugated with MMAE in targeted anticancer therapy

Abstract Background: Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of highly cytotoxic agents, reducing the off-target effects and improve effectiveness with more precising delivery of the payload to the tumor site. Strong correlation has been established between AXL and PD-L1 expression levels in many types of cancer. Therefore, targeting both proteins simultaneously ensure high cancer specificity in delivering the toxic payload. We have developed a bispecific antibody, that strongly induces receptor internalization. Our bispecific ADC combines anti-AXL and anti-PD-L1 construct with toxic MMAE (Monomethyl auristatin E, a potent tubulin inhibitor toxin), for better targeting to solid tumor cells and greatly reducing off-target effects. Materials and Methods: CPL976-MMAE was prepared with site-specific conjugation technology using Fc glycan remodeling and click-chemistry. Briefly, after enzymatic trimming of the N-linked glycans in the Fc pocket of antibody, the terminal GlcNAc was extended with 6-N3-GalNAc using GalNAc-transferase, allowing metal-free click conjugation of the MMAE payload. Bispecific antibodies chosen for conjugation were first characterized in vitro for the interaction with the extracellular domain of human AXL and PD-L1 using surface plasmon resonance and flow cytometry. After the conjugation with MMAE, ADCs cytotoxicity, and selectivity were evaluated in the human breast cancer model (MDA-MB-231) with high expression of both targets, AXL and PD-L1, using SytoX and Hoechst. The human embryonic kidney cell line (HEK-293) with minimal expression of targets, was used as a negative control. The efficacy of conjugates were evaluated in xenograft mouse model. Results: Analytical characterization by RP-HPLC confirmed efficient conjugation and homogeneity of the conjugates. Conjugates showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the AXL/PD-L1 overexpressing cancer cell lines. Additionally, HEK-293 cells were not affected by conjugates in concentrations applied in the assay with use of the breast cancer cell lines. This implies enhanced safety by reducing off-target side effects compared to free auristatin. The efficacy of the compound was confirmed in xenograft mouse model. Conclusions: We have designed a new, potentially first-in-class anti-AXL/PD-L1 bispecific antibody conjugated with MMAE, which showed robust and selective antitumor effects in human cancer models. Citation Format: Delfina Popiel, Krzysztof Lacek, Anna Jabłońska, Aleksandra Sowińska, Agnieszka Bojko - Matuszek, Damian Kołakowski, Filip Mituła, Sebastian Kwiatkowski, Magdalena Bojko, Tomasz Kornatowski, Beata Kliszcz, Tomasz Banach, Michał Górka, Jerzy Pieczykolan, Maciej Wieczorek, Olga Abramczyk. Design and preclinical evaluation of CPL976-MMAE - novel, potent AXL-PD-L1 bispecific antibody conjugated with MMAE in targeted anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B146.

Identification of acetylshikonin as a novel tubulin polymerization inhibitor with antitumor activity in human hepatocellular carcinoma cells.

Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer. Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09-7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation. In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.

Brentuximab Vedotin Combined with Chemotherapy in the Treatment of Pediatric Patients with R/R Hodgkin Lymphoma: Results of a Real World Setting Study

INTRODUCTION: Hodgkin Lymphoma (HL) is a familiar malignant lymphoma of the lymphatic system that accounts for 10% of all lymphoma diagnoses and 5% of lymphoma-related deaths. According to statistics, the incidence of HL in children and adolescents (0-19 years old) in China is about 1.78/million, and an average of 569 new cases are estimated annually from 2019 to 2021 . Brentuximab vedotin (BV) is an antibody-drug conjugates (ADC) that selectively delivers monomethyl auristatin E, an antimicrotubule agent, into CD30-expressing cells. Most of the data on the efficacy and safety of BV came from prospective and retrospective studies in adults or foreign children, and there are insufficient data in Chinese children. The purpose of this study is to explore the efficacy and safety of BV Combined with chemotherapy in Chinese pediatric patients with refractory and/or relapsed Hodgkin's lymphoma. METHODS: Retrospective analysis of clinical data of 9 children with refractory and/or relapsed Hodgkin's lymphoma from October 2021 to May 2023 at Beijing Children's Hospital, Capital Medical University. Patients were classified according to: clinical stage(Ann Arbor stage), huge tumor, huge tumor in the mediastinum, symptoms of B, and whether multiple lymph nodes were involved. Among them, the low-risk group was IA, ⅡA without giant tumor. The middle risk group was IB and ⅢA without huge tumor. High-risk groups were patients with stage ⅡB, ⅢB, Ⅳ or (each stage) with large tumors, or children with > 4 lymph node regions. All patients received BV in combination with chemotherapy for at least 2 cycles, and patients were assessed for remission response according to the Revised Response Criteria for Malignant Lymphoma at the end of every 2 cycles and followed up until July 20, 2023. RESULTS: Totally 32 cycles of chemotherapy were recorded, including 36 doses of BV administration(Table 1). The overall response rate of the children after 2 cycles of treatment was 100%, and the complete response rate was 88.89%. After remission, 2 cases (P1, P2) were treated with BV single drug 1.8mg/kg, Q3W for 6 months, and 3 cases (P3, P4, P7) were treated with radiotherapy. Two patients (P5, P6) received sequential chemotherapy, and two patients (P8, P9) were followed by programmed cell death 1(PD-1) antibody maintenance treatment for 6 months. After receiving PD-1 antibody for 6 months, 1 patient (P9) was transferred to BV monotherapy for maintenance, with 18 times planned. 2 cases (P1, P2) were then treated with autologous stem cell transplantation. None of the children developed disease progression during treatment. All patients successfully completed all planned dosage of BV, and serious adverse events were dominated by grade 3 or higher hematocrit, none of which interfered with normal treatment. Interpretation. BV had demonstrated the better efficacy and tolerable safety profile in the treatment of refractory and/or relapsed Hodgkin's lymphoma in children. CONCLUSIONS: BV has been approved by National Medical Products Administration in China since 2020, the clinical cases are mostly from adult lymphoma patients, and children data of in China is rare. In this study, we retrospectively reviewed and analyzed the data of BV combined chemotherapy in the treatment of refractory and relapsed Hodgkin lymphoma from single center, and found that the treatment response was rapid, continuousand safe. However, this study is a single-center retrospective study with a small sample size So We are conducting a prospective study of BV in first-line Hodgkin lymphoma children patients in China and aiming to generate more data for this specific population.

Development of First in Class DNA-Zip Code Drug Conjugate (ZDC) in Multiple Myeloma

Multiple myeloma is a heterogeneous malignant plasma cell disorder with complex molecular and genetic abnormalities. Significant advances have been made in the treatment of multiple myeloma; however, a large proportion of patients fail to attain a favorable response and relapse. Due to efficacy and toxicity, the development of drug conjugates for multiple myeloma has been limited. Our lab discovered that circulating tumor DNA (ctDNA) serves as a cell-specific mechanism for genetic transmission. Certain retrotransposon sequences within the ctDNA were identified as being crucial for the tropism of ctDNA to target tumor cells (Cinar et al bioRxiv.2022). We therefore query whether these transposons can be exploited for the targeted drug delivery. To do so, chemically synthetize and CY5-labeled multiple retrotransposons DNA sequences (a.k.a Zip Codes, ZC) to perform high through put screening assessing for cell targeting. Our findings showed that the AluSp and MER11c DNA sequences (MM-ZC1 and 2) with unique MM mutations were the most efficient and specific DNA sequences for targeting myeloma cells. Furthermore, Flow cytometry and CYTOF validated the specific MM cell targeting on full bone marrow samples from MM patients. Based on this analysis we used MMZC1 for further drug development. We design a drug conjugation approach for the MMZC and screen various drugs for their killing potential. Two potent MM ZC drug conjugates (ZDC) were identified by high throughput viability screening assay: ZC-DM4, a microtubule inhibitor, and ZC-Carfilzomib, an irreversible proteasome inhibitor. We move forward exploration with ZC-DM4 due to limited carfilzomib-linker drug availability. Experiments cancer cell lines in vitro demonstrated that MMZC-DM4 had a potent killing effect on MM cells (IC50: 3.25 nM) but not on other cancer cell lines. These results were the validated in vivo. Maximum tolerated dosing studies (MTD) demonstrated excellent tolerability in all doses (ranges from 1 to 20 mg of MMZC-DM4/Kg (equivalent to 0.007-0.149 mg of DM4/kg). To look for an efficacy signal of MMZC-DM4, MM xenograft mice were given MMZC-DM4 twice a week for three weeks. Due to limitations in production, we treated the animals biweekly with three distinct low doses of MMZC-DM4: 1.9, 3.75, and 7.5 mg/kg (equivalent to 0.014, 0.028, and 0.056 mg of DM4/kg) and compared their response to mice not treated with MMZC-DM4 and mice treated weekly with bortezomib. MMZC-DM4 at such small doses reduced the tumor by 30% without affecting body weight, WBC, HB, AST, or platelets. In contrast, Bortezomib was associated with 90% growth inhibition at the expense of significant weight loss that required treatment interruptions and thrombocytopenia. Currently, investigations are being conducted to examine the therapeutic efficacy of greater doses of MMZC-DM4. This study described the first-in-class MM targeted medicine delivery employing an MM zic code drug conjugate. Our findings suggest new method for targeted delivery therapeutic cargo material directly to cancer cells that could revolutionize cancer treatment.

Measuring Drug Response with Single-Cell Growth Rate Quantification.

Intratumoral heterogeneity is a substantial cause of drug resistance development during chemotherapy or other drug treatments for cancer. Therefore, monitoring and measuring cell exposure and response to drugs at the single-cell level are crucial. Previous research suggested that the single-cell growth rate can be used to investigate drug-cell interactions. However, currently established methods for quantifying single-cell growth are limited to isolated or monolayer cells. Here, we introduce a technique that accurately measures both 2D and 3D cell growth rates using label-free ratiometric stimulated Raman scattering (SRS) microscopy. We use deuterated amino acids, leucine, isoleucine, and valine, as tracers and measure the C-D SRS signal from deuterium-labeled proteins and the C-H SRS signal from unlabeled proteins simultaneously to determine the cell growth rate at the single-cell level. The technique offers single-cell level drug sensitivity measurement with a shorter turnaround time (within 12 h) than most traditional assays. The submicrometer resolution of the imaging technique allows us to examine the effects of chemotherapeutic drugs, including kinase inhibitors, mitotic inhibitors, and topoisomerase II inhibitors, on both the cell growth rate and morphology. The capability of quantifying 3D cell growth rates provides insight into a deeper understanding of the cell-drug interaction in the actual tumor environment.

Zilovertamab Vedotin (MK-2140) in Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL): Updated Results from the Phase 2 Waveline-004 Study

Background: DLBCL is an aggressive non-Hodgkin lymphoma (NHL) that is often curable with frontline therapy, but up to 50% of patients (pts) will develop relapsed or refractory (R/R) disease. Treatment options are limited for pts with R/R DLBCL who have progressed after or are ineligible for autologous stem cell transplant (ASCT) or chimeric antigen receptor (CAR) T-cell therapy. ROR1 is an oncofetal transmembrane protein that is expressed in DLBCL. Zilovertamab vedotin, an antibody-drug conjugate comprising a humanized monoclonal antibody against ROR1, a proteolytically cleavable linker, and the antimicrotubule agent monomethyl auristatin E (MMAE), initially demonstrated promising antitumor activity and manageable safety in R/R NHL in the phase 1 waveLINE-001 study. Early results from the single-arm, open-label, phase 2 waveLINE-004 study (NCT05144841) showed that zilovertamab vedotin had clinically meaningful antitumor activity and manageable safety in pts with R/R DLBCL who progressed after or were ineligible for ASCT and/or CAR T-cell therapy. We present updated results from waveLINE-004. Methods: Eligible pts were ≥18 y, had histologically confirmed R/R DLBCL per WHO classification, measurable disease per Lugano 2014 criteria, PET-positive disease, and an ECOG performance status of 0 to 2. Pts were required to have disease progression after ≥2 prior lines of therapy including an alkylating agent, anthracycline, and an anti-CD20 antibody, and had progressed after or were ineligible for ASCT and CAR T-cell therapy. Pts received zilovertamab vedotin 2.5 mg/kg IV Q3W until disease progression, unacceptable toxicity, or withdrawal. Primary end point was ORR per Lugano 2014 criteria. Secondary end points were DOR and safety and tolerability. PFS and OS were exploratory. Efficacy is reported for pts who had ≥1 postbaseline assessment available. Safety is reported for all pts who received ≥1 dose of zilovertamab vedotin. Results: At the data cutoff (April 26, 2023), 98 pts had been enrolled and received ≥1 dose of zilovertamab vedotin; 61 pts (62%) had discontinued treatment predominantly because of disease progression (38%); 37 pts (38%) were ongoing. Median age was 66 y (range, 19-88), 63 pts (64%) were male, and 86 (92%) had an ECOG performance status of 0 or 1. Most pts (n = 70; 71%) had received ≥3 prior lines of therapy. Forty-one (42%) and 13 pts (13%) had high-intermediate and high-risk NCCN-IPI scores, respectively. Fifteen pts (15%) had undergone prior ASCT, 18 (18%) had undergone prior CAR T-cell therapy, and 5 (5%) had undergone both ASCT and CAR T-cell therapy. Median time from first dose to data cutoff was 4.5 mo (range, 0.2-13.2) for pts who received ≥1 dose of zilovertamab vedotin, and 5.6 mo (range, 0.9-13.2) for the 79 pts who had ≥1 postbaseline scan (efficacy analysis population). ORR per investigator review was 29% (95% CI, 19.4-40.4), with 10 pts (13%) having a complete response and 13 (16%) a partial response; 10 pts (13%) had stable disease, for a disease control rate of 42% (95% CI, 30.8-53.4). Median DOR was 3.0 mo (range, 0.0+ to 8.8+). Median PFS was 2.5 mo (95% CI, 1.9-3.0); 6-mo PFS was 15%. Median OS for all 98 pts was 10.6 mo (95% CI, 5.1-NR); 6-mo OS was 62%. Treatment-related AEs occurred in 78 pts (80%), most commonly (≥20%) neutrophil count decreased (n = 47; 48%), anemia (n = 25; 26%), diarrhea (n = 23; 23%), and white blood cell count decreased (n = 20; 20%). Grade 3-5 treatment-related AEs occurred in 51 pts (52%), most commonly (≥10%) neutrophil count decreased (n = 38; 39%), anemia (n = 15; 15%), and white blood cell count decreased (n = 12; 12%). Two pts (2%) died because of treatment-related AEs (septic shock, acute kidney injury). Four pts (4%) discontinued zilovertamab vedotin because of treatment-related AEs (2 diabetic ketoacidosis, 1 septic shock, 1 acute kidney injury). Peripheral neuropathy occurred in 18 pts (18%), 2 (2%) of whom had a grade 3/4 event. Dose modification because of peripheral neuropathy was required for 10 pts (10%). One pt (1%) experienced a grade 2 treatment-related infusion reaction. Conclusions: In this updated analysis of waveLINE-004, zilovertamab vedotin continued to demonstrate clinically meaningful antitumor activity in pts with R/R DLBCL who had progressed after or were ineligible for ASCT and/or CAR T-cell therapy. The safety profile of zilovertamab vedotin was manageable, and consistent with the known profile of MMAE-containing agents.

Efficacy and Safety of Polatuzumab Vedotin in Follicular Lymphoma: A Systematic Review of Clinical Trials

Introduction: Polatuzumab vedotin (Pola) is a humanized anti-CD79b monoclonal antibody linked with antimitotic agent monomethyl auristatin E. On internalization, the linker is cleaved, and antimitotic activity inhibits the cell division and growth of cancer cells. Pola is tested in multiple combinations in relapsed/refractory (RR) follicular lymphoma (FL) in recent years. In this systematic study, we will assess the efficacy and safety of Pola based combinations in RR FL. Methods: A literature search was performed on PubMed with keywords, “Polatuzumab vendotin” and “lymphoma” from the inception of data till 07/01/2023. PRISMA guidelines were followed while conducting this systematic review. We reviewed 160 articles and included 1 randomized clinical trial (RCT, N=41), and 4 non-randomized clinical trials (nRCT, N=163) based on inclusion criteria. Data was extracted for complete response (CR), partial response (PR), overall response (ORR), progression free survival (PFS), overall survival (OS) and ≥grade 3 side effects. Results: In 5 trials (N=204), 13 patients with Pola + atezolizumab (Ate) + obinutuzumab (Obi), 49 patients with Pola + Obi + venetoclax (Ven), 39 patients with Pola + Obi, 62 with Pola + Obi + lenalidomide (Len), and 20 patients with Pola + rituximab (Rit). In an RCT by Morschhauser et al. (N=41), Pola + Rit was compared with pinatuzumab vedotin (Pina) + Rit. ORR, CR, PR, and mPFS was 70%, 45%, 25%, and 15.3 months, respectively with Pola + Rit, as compared to 62%, 5%, 57%, and 12.7 months, respectively, with Pina + Rit. In a trial by Philips et al. (N=39), ORR, CR, PR, and mPFS were 67%, 36%, 31%, and 11.5 months, respectively. In 3 nRCTs (N=124), three drug regimens were used with Pola and Obi as backbone. ORR, CR, PR were 50%-83%, 20%-61%, and 20%-39%. Table 1. ≥ Grade 3 hematological and non-hematological side effects are given in table 1. Conclusion: Pola was well tolerated by most of the patients with FL alone or in combination with Obi, Rit, Len, Ven. Pola had relatively better response and survival rates as compared to Pina in combination with Rit. In early phase clinical trials, three drug regimens of Pola and Obi with Ven, and Len were effective in the treatment of heavily pretreated follicular lymphoma. In an early phase trial, Pola + Obi + Ate had limited efficacy in FL with safety concerns. More randomized double blind multicenter phase III clinical trials are needed to confirm these results.

Discovery of a Dual Tubulin and Neuropilin-1 (NRP1) Inhibitor with Potent In Vivo Anti-Tumor Activity via Pharmacophore-based Docking Screening, Structure Optimization, and Biological Evaluation.

Dual inhibition of tubulin and neuropilin-1 (NRP1) may become an effective method for cancer treatment by simultaneously killing tumor cells and inhibiting tumor angiogenesis. Herein, we identified dual tubulin/NRP1-targeting inhibitor TN-2, which exhibited good inhibitory activity against both tubulin polymerization (IC50 = 0.71 ± 0.03 μM) and NRP1 (IC50 = 0.85 ± 0.04 μM). Importantly, it significantly inhibited the viability of several human prostate tumor cell lines. Further mechanism studies indicated that TN-2 could inhibit tubulin polymerization and cause G2/M arrest, thereby inducing cell apoptosis. It could also suppress cell tube formation, migration, and invasion. Moreover, TN-2 showed obvious antitumor effects on the PC-3 cell-derived xenograft model with negligible side effects and good pharmacokinetic profiles. These data demonstrate that TN-2 could be a promising dual-target chemotherapeutic agent for the treatment of prostate cancer.

Genome-wide CRISPR/Cas9 screening for drug resistance in tumors

Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) screening is a simple screening method for locating loci under specific conditions, and it has been utilized in tumor drug resistance research for finding potential drug resistance-associated genes. This screening strategy has significant implications for further treatment of malignancies with acquired drug resistance. In recent years, studies involving genome-wide CRISPR/Cas9 screening have gradually increased. Here we review the recent application of genome-wide CRISPR/Cas9 screening for drug resistance, involving mitogen-activated protein kinase (MAPK) pathway inhibitors, poly (ADP-ribose) polymerase inhibitors (PARPi), alkylating agents, mitotic inhibitors, antimetabolites, immune checkpoint inhibitors (ICIs), and cyclin-dependent kinase inhibitors (CDKI). We summarize drug resistance pathways such as the KEAP1/Nrf2 pathway MAPK pathway, and NF-κB pathway. Also, we analyze the limitations and conditions for the application of genome-wide CRISPR/Cas9 screening techniques.

Synthesis, Characterization and Biological Evaluation of New Enamide Fluorinated‐Schiff Base Derivatives as Potential Cytotoxic and Apoptosis‐Inducing Agents

AbstractIn search for new anticancer candidates, a new series of enamide fluorinated‐Schiff base derivatives were synthesized and confirmed by spectroscopic techniques as well as elemental microanalyses. The newly prepared compounds were evaluated for their cytotoxic activity on MCF‐7 breast adenocarcinoma cell line. The benzaldehyde Schiff base and furfural Schiff base derivatives (IC50=3.67±0.15 and 2.06±0.08 μM, respectively) were found to be the most potent molecules of all the tested derivatives against the MCF‐7 cancer cells. Additionally, the furfural Schiff base compound was more potent than colchicine by 1.74‐fold, while benzaldehyde Schiff base compound was nearly equipotent with colchicine as standard drug. In addition to tubulin inhibition activity, enamide‐furfural Schiff base molecule caused cell cycle arrest in MCF‐7 cells at the G2/M phase (2.62‐fold more than control MCF‐7) and induced apoptotic death (48.97‐fold compared to untreated MCF‐7 control) as indicated by FACS analysis. Furthermore, apoptotic studies conducted by enamide‐furfural Schiff base derivative were accomplished by downregulation of mitochondrial membrane potential by 1.56‐fold relative to control untreated cells.

MitoCur-1 induces ferroptosis to reverse vemurafenib resistance in melanoma through inhibition of USP14.

Melanoma is an aggressive malignant tumor with a poor prognosis. Vemurafenib (PLX4032, vem) is applied to specifically treat BRAF V600E-mutated melanoma patients. However, prolonged usage of vem makes patients resistant to the drug and finally leads to clinical failure. We previously tested the combination regimen of tubulin inhibitor VERU-111 with vem, as well as USP14 selective inhibitor b-AP15 in combination with vem, both of which have showed profound therapeutic effects in overcoming vem resistance invitro and invivo. Most importantly, we discovered that vem-resistant melanoma cell lines highly expressed E3 ligase SKP2 and DUB enzyme USP14, and we have demonstrated that USP14 directly interacts and stabilizes SKP2, which contributes to vem resistance. These works give us a clue that USP14 might be a promising target to overcome vem resistance in melanoma. MitoCur-1 is a curcumin derivative, which was originally designed to specifically target tumor mitochondria inducing redox imbalance, thereby promoting tumor cell death. In this study, we have demonstrated that it can work as a novel USP14 inhibitor, and thus bears great potential in providing an anti-tumor effect and sensitizing vem-resistant cells by inducing ferroptosis in melanoma. Application of MitoCur-1 dramatically induces USP14 inhibition and inactivation of GPX4 enzyme, meanwhile, increases the depletion of GSH and decreases SLC7A11 expression level. As a result, ferrous iron-dependent lipid ROS accumulated in the cell, inducing ferroptosis, thus sensitizes the vem-resistant melanoma cell. Interestingly, overexpression of USP14 antagonized all the ferroptosis cascade events induced by MitoCur-1, therefore, we conclude that MitoCur-1 induces ferroptosis through inhibition of USP14. We believe that by inhibition of USP14, vem resistance can be reversed and will finally benefit melanoma patients in future.

Programmed initiation and enhancement of cGAS/STING pathway for tumour immunotherapy via tailor-designed ZnFe2O4-based nanosystem.

The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signalling pathway has been a promising target for anticancer immunity, but rationally activating and enhancing this pathway in tumour cells is critical. Herein, a glutathione sensitive ZnFe2O4-based nanosystem is developed to programmatically initiate and enhance the STING signalling pathway in tumour cells. The prepared ZnFe2O4 nanoparticles were coated with cancer cell membrane (CCM), which enabled the nanosystem target tumour cells. In tumour cells, ZnFe2O4 nanoparticles could be disintegrated by responding to high level glutathione, and the released Fe3+ generated reactive oxygen species to induce the DNA leakage into the cytoplasm to stimulate cGAS. Then Zn2+ promoted cGAS-DNA phase separation to intensify the cGAS enzymatic activity. In addition, the low dose encapsulation of paclitaxel (PTX) acting as an antimitotic agent (ZnFe2O4-PTX@CCM) ensured the sustained activation of cGAS/STING pathway. The in vitro and in vivo results confirmed that ZnFe2O4-PTX@CCM elevated the cGAS/STING activity, promoted dendritic cell maturation, increased cytotoxic T lymphocyte and natural killer cells infiltration, eventually inhibiting the tumour progress and postoperative recurrence. This study provided feasible references on constructing STING activation nanosystem for tumour immunotherapy.

RBIO-05. MITOTIC ENRICHMENT AS AN EFFICIENT RADIOSENSITIZATION STRATEGY FOR INTRACRANIAL TUMORS

Abstract Radiotherapy remains one of the most effective modalities for anticancer treatment and part of standard of care for many primary and metastatic brain tumors. Boosting the efficacy of radiotherapy is therefore a logical avenue to improve patient survival. We have developed a radiosensitization strategy called ‘induction of mitotic enrichment’. It has long been known that the radiosensitivity of a cell depends on the phase of the cell cycle and that mitotic cells are especially vulnerable. Enriching the tumor for mitotic cells by arresting them during division prior to each radiotherapy fraction should therefore render the tumor population more sensitive to irradiation. Ideally, induction of mitotic enrichment should be reversible and non-cytotoxic to prevent healthy tissue toxicity and be compatible with clinically applied fractionated radiotherapy regimens. We have now identified an orally available targeted tubulin polymerization inhibitor that can achieve repeated and reversible mitotic enrichment for up to 10 hours prior to radiotherapy, without causing cytotoxicity in vitro or healthy tissue toxicity in vivo. This tubulin inhibitor (ABT-751) efficiently radiosensitizes a range of preclinical tumor models representing adult and pediatric, primary and metastatic brain tumors. Importantly, ABT-751 also improves survival in mouse models of adult glioblastoma (GBM), but only in a mitotic enrichment setup when given several hours prior to radiotherapy to allow accumulation in mitosis. Of note, induction of mitotic enrichment does not exacerbate healthy tissue toxicity. To conclude, induction of mitotic enrichment by ABT-751 radiosensitizes adult GBM to fractionated radiotherapy. We are currently expanding our preclinical development of mitotic enrichment as a radiosensitization strategy to other targets and cancers and have designed a phase 0/I trial for adult GBM to demonstrate induction of mitotic enrichment and tolerability when combined with radiotherapy. We expect to start enrollment in this trial soon.

Performance of the FACT-GOG-Ntx to assess chemotherapy-induced peripheral neuropathy (CIPN) in pediatric high risk Hodgkin lymphoma: report from the Children’s Oncology Group AHOD 1331 study

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) is an under-recognized complication of several chemotherapy agents used as part of curative-intent therapy for Hodgkin Lymphoma (HL). In the absence of validated self- or proxy-report measures for children and adolescents, CIPN reporting has relied on clinician rating, with grading scales often restricted to severe manifestations. In a proof-of-concept study, we assessed the feasibility and psychometric performance of the Functional Assessment of Cancer Therapy-Gynecologic Oncology Group-Neurotoxicity (FACT-GOG-Ntx), a unidimensional CIPN symptom scale widely used adults with CIPN, in pediatric HL at risk for CIPN.MethodsYouth (11+ years) and parents of all children (5–17.9 years) with newly diagnosed high-risk HL enrolled on Children’s Oncology Group AHOD1331 (NCT02166463) were invited to complete the FACT-GOG-Ntx and a health-related quality of life (HRQL) measure at pre-treatment (Time 1), and during cycles 2 (Time 2) and 5 (Time 3) of chemotherapy during the first half of study accrual. Clinical grading of CIPN by providers was also assessed using the Balis Pediatric Neuropathy Scale. We evaluated Cronbach’s alpha, construct validity, and agreement between raters. Change in FACT-GOG-Ntx scores over time was assessed using a repeated measures model.Results306 patients had at least one completed FACT-GOG-Ntx with time-specific completion rates of > 90% for both raters. Cronbach’s alpha was > 0.7 for youth and parent-proxy report at all time points. Correlations between FACT-GOG-Ntx and HRQL scores were moderate (0.41–0.48) for youth and parent-proxy raters across all times. Youth and parent-proxy raters both reported worse FACT-GOG-Ntx scores at Time 3 for those who had clinically-reported CIPN compared to those who did not. Agreement between raters was moderate to high. Compared to baseline scores, those at Time 3 were significantly lower for youth (β = − 2.83, p < 0.001) and parent-proxy raters (β = − 1.99, p < 0.001).ConclusionsHigh completion rates at all time points indicated feasibility of eliciting youth and parent report. Psychometric performance of the FACT-GOG-Ntx revealed acceptable reliability, evidence of validity, and strong inter-rater agreement, supporting the use of this self- or proxy-reported measure of CIPN in youth with high-risk HL exposed to tubulin inhibitors, as part of a Phase 3 clinical trial.Clinical trial information: Clinical Trials Registry, NCT02166463. Registered 18 June 2014, https://clinicaltrials.gov/ct2/show/study/NCT02166463