Mcl-1 Expression Research Articles

Correlation of Flow Cytometric BCL2 and MCL1 Expression with Cytogenetic Characteristics and Outcome in Multiple Myeloma

Introduction: Clonal plasma cells mediate apoptosis resistance through increased expression of anti-apoptotic proteins which increase survival and mediate resistance to intrinsic mitochondrial pathway. The key anti-apoptotic members are BCL-2, MCL-1, BCL-XL, BCL-B and BCL-W. High BCL2 expression or t(11;14) is shown to be predictive of response to Venetoclax in Multiple Myeloma (MM). Venetoclax may only be effective in a subset of patients, namely those who have relatively high BCL2 and low MCL1. Resistance to Venetoclax can be mediated by mutation in BCL2 or gain in copy number of MCL1, which maps to 1q21. The relationship between cytogenetic abnormalities and expression of BCL2 and MCL1 is not clear except for an occasional study which has shown MM with CCDN1 translocation has high BCL2 and low MCL1 expression and MM with MMSET and MAF translocation has high MCL1 and low BCL2 expression. Also, previous studies have used either immunohistochemistry or real time quantitative PCR for studying BCL2 and MCL1 expression, both of which are met with challenges in interpretation. We aimed to correlate the intracellular expression of BCL2 and MCL1 by flow-cytometry (FCM) with cytogenetic characteristics and outcome. Methodology: FCM was performed in bone-marrow of 143 consecutive MM cases with ≥10% plasma cells (PCs) and 20 bone marrow samples with non-clonal PCs by lyse-stain-wash method followed by intracellular staining of BCL2 (Alexa Fluor 647 conjugated anti-human BCL2 antibody) and MCL-1 (PE-conjugated anti-human MCL1 antibody). The sequential gating strategy used in the analysis of intracellular BCL2 and MCL1 expression in PCs and T lymphocytes is shown in Figure 1. The percentage of PCs positive for BCL2 and MCL1 compared to the tube without these antibodies [Fluorescence Minus One (FMO) cutoff threshold], the mean fluorescence intensity (MFI) expression of BCL2 and MCL1 in PCs and T cells, and the ratio of expression of BCL2 and MCL1 on PCs and T cells (BCL2 PC/T, MCL1 PC/T), MFI BCL-2 PC/MFI MCL1 PC were studied and further correlated with 5-7 panel FISH cytogenetics by using Kruskal Wallis test or Mann-Whitney U test. The overall survival/OS and progression-free survival/PFS was calculated using Kaplan-Meir Survival Curve analysis. Result and discussion: During the study, FCM was performed in 143 MM patients (103 Newly Diagnosed MM and 40 Relapsed Refractory MM). The cytogenetic findings includes IGH:: FGFR3 (n=29), IGH:: CCND1 (n=8), IGH:: MAF (n=3), gain1q21 (n=76), 1q21-amplification (n=30), 1p32-deletion (n=17) and TP53-deletion (n=21). A median of 38,779 PCs was acquired by FCM. The BCL2 and MCL1 expression was seen in 4-100% (median-96.9%) and 0-100% (median-78.8%) of clonal PCs, respectively. The median BCL2 MFI and MCL1 MFI were 43,289 and 6,481 respectively. The median BCL2 PC/T, MCL1 PC/T and BCL2 PC/MCL1 PC in clonal PCs were 1.90, 4.35, and 07.1, respectively, whereas in non-clonal PCs they were 0.87, 3.02 and 3.21, respectively.BCL2 PC/T, MCL1 PC/T and BCL2 PC/MCL1 P were significantly higher in clonal PCs than non-clonal PCs, with a p-value of 0.002, 0.001, and 0.009, respectively. The median MCL1 MFI was significantly lower (3,825 vs. 6,559; p=0.020) and median BCL2 PC/MCL-1 PC (12 vs 6.7; p=0.012) was significantly higher in IGH:: CCND1 compared to others; while an opposite pattern was seen in gain1q21 (7,420 vs 5,525; p=0.032 and 5.5 vs 8.6; p=0.046). The dim expression (<25-percentile) of BCL2 and MCL1 was noted in 25% (n=36) MM cases. Survival analysis did not yield a significant difference in OS or PFS between patients with dim, moderate or strong expression of BCL2 or MCL1. Conclusions: Our study demonstrates an objective assessment of BCL2 and MCL1 expression by FCM. BCL2 PC/T,MCL1 PC/T and BCL2 PC /MCL1 PC were significantly higher in myeloma cells compared to non-clonal PCs. Low MCL1 expression or high BCL2/MCL1 ratio was associated with IGH:: CCND1; while strong MCL1 and low BCL2/MCL1 ratio was most frequent in 1q21 amplification. We identified dim-expression (< 25-percentile) of BCL2 or MCL1 in 25% of cases of MM, where the benefit of anti-BCL2/MCL1 targeted therapy needs to be studied. Routine testing of BCL2 and MCL1 expression by FCM may be beneficial before starting Venetoclax/similar targeted therapy.

Targeting G3BP2 Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment Via ELF1-MCL1 Axis

Venetoclax-based combination therapies have emerged as promising treatments for elderly acute myeloid leukemia (AML) patients. However, venetoclax resistance renders the treatment ineffective, mainly due to the upregulation of MCL1. Thus, new venetoclax-based combination therapy is under development to circumvent venetoclax resistance. The Ras-GTPase-activating protein SH3-domain binding protein 2 (G3BP2) is upgraded in several solid tumors. Targeting G3BP2 re-sensitized tumor cells to chemotherapy. To date, whether targeting G3BP2 sensitizes venetoclax treatment in AML and its role in leukemogenesis has not been defined. To this end, the primary AML cells and AML cells were collected and cultured with compound C108 (G3BP2 inhibitor) and venetoclax alone or both. The result showed that co-culture with compound C108 and venetoclax decreased the cell viability in primary AML and AML cells, indicating the potent synergy of G3BP2 inhibitor and venetoclax. The Quantitative real-time PCR (RT-qPCR) showed that G3BP2 was overexpressed in AML patients and was associated with poor prognosis. Knockdown (KD) of G3BP2 decreased proliferation and induced apoptosis in AML cells. In addition, G3BP2 inhibition impeded the tumorigenicity in the subcutaneous AML xenograft tumor model. These data demonstrated that G3BP2 sensitized AML to venetoclax treatment and is critical in AML development. The RNA-seq analysis was performed to investigate the role of G3BP2 in venetoclax resistance and leukemogenesis. The results showed that differentially expressed genes (DEG) were enriched in apoptotic processes when G3BP2 were downregulated. The immunoblotting results indicated that G3BP2 knockdown decreased the expression of MCL1 and BCL2, and vice versa. Moreover, we found that the transcription factor ELF1 was positively correlated with G3BP2 by the immunoblotting and RNA-seq analysis. The result was further validated by the Pearson correlation analysis based on the online database. The expression of ELF1 was decreased when G3BP2 were downregulated. Thus, we concluded that G3BP2 is an upstream gene of ELF1 and MCL1. To further investigate the relationship among G3BP2, ELF1, and MCL1, the RNA binding protein immunoprecipitation assay (RIP) and mRNA stability analysis was performed. The results suggested that G3BP2 interacted with the ELF1 mRNA to enhance its stability. We employed bioinformatics analysis via the JASPAR database and RPISeq tools and found ELF1 may facilitate the transcription of MCL1, which was validated in the following study. The expression of MCL1 was restored in the G3BP2 KD cells when ELF1 was overexpressed. We subsequently performed CHIP-qPCR, dual-luciferase reporter gene assays with truncated MCL1 promoter fragments. A 576bp of the binding region upstream of MCL1 promoter was defined, indicating that ELF1 regulated MCL1 expression by modulating its transcription activity. Taken together, our data suggested that targeting G3BP2 decreases MCL1 expression by disturbing ELF1 mRNA stability, thus increasing the sensitivity to venetoclax treatment. We finally established the intra-venous tumorigenesis xenograft model to verify the role of G3BP2 downregulation in venetoclax-treated leukemia mice. The NCG immunodeficient mice were transplanted with U937 cells and administrated with venetoclax daily from 7-21 days post transplantation (Figure 1A). The data showed that G3BP2 KD in combination with venetoclax reduced clinical scores and prolonged the overall survival of AML mice (combination vs. venetoclax: p<0.01; combination vs. G3BP2 KD: p<0.05, Figure 1B). The reduced leukemic burden was observed when targeting G3BP2 in combination with venetoclax. Meanwhile, we verified the decreased expression of ELF1 and MCL1 in mice targeting G3BP2 in combination with venetoclax by immunofluorescence (IF) and flow cytometry analysis. These data supported the synergic effect of G3BP2 inhibition and venetoclax treatment in AML. Our findings emphasize the critical role of G3BP2 in conferring resistance to venetoclax in AML by stabilizing ELF1 mRNA to regulate MCL1 expression. Targeting G3BP2 represents an appealing therapeutic strategy for patients with venetoclax-resistant AML.

Significance of the Apoptotic Regulators BAX, BCL2, BCL- XL and MCL1 in Newly Diagnosed AML

Introduction: In acute myeloid leukemia (AML), cytogenetics and molecular mutations remain important prognostic tools for tailoring treatment after induction therapy. Abnormalities in multiple components of apoptotic pathways have been identified as potential drivers of AML. Identifying other biomarkers may help us further refine prognosis and identify other therapeutic strategies. B-cell lymphoma 2 (Bcl2), Bcl-xL and myeloid cell leukemia-1 (Mcl1) are commonly expressed prosurvival proteins in hematologic malignancies, whereas Bax acts as a promoter of apoptosis. We retrospectively evaluated the significance of Bax, Bcl2, Bcl-xL and Mcl1 expression in newly diagnosed AML patients using the Beat AML program 1. Methods: We analyzed RNA-expression data from The Beat AML study (672 tumor specimens collected from 562 patients) 1. Data was extracted from the public database. Different expression levels of the above genes were compared using t-tests. Results were considered significant with p-value < .05. Kaplan-Meier curves were used to estimate survival. Results: For the correlative survival analyses, Bax, Bcl2, Bcl-xL and Mcl1 mRNA expression and clinical information were both obtained from the BeatAML cohort 1. Patients were categorized into high expressers (≥ median) and low expressers (<median). In total, 461 patients had available gene expression data. The mean age of diagnosis was 57 years, and 56.6% of patients were male. The most common specific diagnosis at inclusion was AML with mutated NPM1 (22%), followed by AML with myelodysplasia related changes (21%) and AML, NOS (11%). Complete response to induction was seen in more than half (56%) of the patients for which response was recorded (n=369). Others developed refractory AML (32%). No data was available for 12%. Standard chemotherapy (induction therapy with idarubicin and cytarabine (7+3) and consolidation therapy with high dose cytarabine) was used in 98.4% of these patients. Over half of the analyzed samples were peripheral blood (52%), and the remaining samples were either bone marrow aspirate (46%) or from leukapheresis (2%). Most of the specimens were collected at initial acute leukemia diagnosis (62.2%), others were collected with residual disease or post chemotherapy (25.1%). The mean survival for this cohort was 13.7 months. Above median expression of Mcl1 was associated with longer survival (33 vs. 20 months; p = 0.0262). Above median expression of Bax was also associated with longer survival (32 vs. 18.2 months; p = 0.0114). In the Bax analysis, gene expression was further subdivided into 4 quartiles which showed correlation of expression levels with length of survival (Figure 1). The prognostic significance of the ratio Bax/ (Mcl1+Bcl2+Bcl-xL) was also analyzed given the mechanism of action of these various regulators. A higher ratio was associated with longer survival (40.7 vs. 18.7 months; p = 0.0095) (Figure 2) Conclusions Bcl-2, Bcl-xL and Mcl-1 are commonly expressed antiapoptotic proteins, whereas Bax is a promoter of apoptosis. The results with Bax are consistent with previous literature. Our results with Mcl1 expression differed from what we may have expected. This may be due to the sample size or methodology. However, it may also be due to the fact that a ratio of the actors is needed to help predict prognosis (as we analyzed in Figure 2). The overall survival between Bcl-2 and Bcl-xL was compared independently, however, they did not appear to have a significant difference in survival between high and low expression groups. We are currently working to see if this ratio can be validated in a prospective cohort of relapsed/ refractory AML patients treated on clinical trial and this data will be presented at the meeting. The Bcl-2 inhibitor venetoclax has demonstrated promising results in treating AML. However, venetoclax appears to have limited activity as a single agent. Our study suggests that potentially a combination of agents targeting factors such as Bcl-2, Bcl-xL and Mcl-1 may be associated with improved survival in patients with AML. Further validation of these biomarkers is warranted, and Mcl-1 inhibitors are currently in clinical trials.

Inhibition of Sphingosine Kinase 2 Enhances Immunotherapy in Mouse Model of Multiple Myeloma

Introduction: Sphingolipid metabolism, in particular the ceramide:sphigosine 1-phophate (S1P) rheostat, is increasingly recognized as a key pathway in cancer biology, inflammation and immune responses. Sphingosine kinase (SK) 1 and 2 catalyze the formation of S1P. We previously reported that SK2 but not SK1 was overexpressed in multiple myeloma (MM) cells. Treatment with opaganib, an SK2-specific inhibitor, downregulated the expression of c-Myc and Mcl-1, resulting in apoptosis of MM cells. A phase I clinical trial of opaganib in patients with relapsed/refractory MM demonstrated the safety and evidence of anti-myeloma activity of SK2 inhibition. However, the roles of SK2 in host's anti-tumor immunity are unclear. We herein determined the effects of SK2 on the functions of T cells and myeloid derived suppressor cells (MDSCs), the underlying mechanisms, and potential clinical application of SK2 inhibition in enhancing immunotherapy. Methods: For tumor models, transplantable mouse VK*Myc myeloma cells, CT-2A gliomas cells, B16F10 melanoma cells, or TRAMP C2 prostate cancer cells were implanted into SK2 -/- knockout (KO), SK1 -/- KO or WT mice. Multi-color flow cytometry was used to measure various immune cells. Anti-CD8 antibody (clone YST-169.4) and anti-Gr-1 antibody (clone RB6-8C5) were used to deplete CD8 T cells and MDSCs in vivo, respectively. Adaptive transfer of enriched CD8 T cells and bone marrow derived MDSCs was performed. Cytokines and chemokines were measured by multiplex assay. Transcriptome sequencing and omics analyses were performed on mouse primary CD8 T cells and MDSCs. CRISPR/Cas9 genetic knockout experiments were used to validate genes identified from the sequencing results. Mouse BCMA CAR T model was established using syngeneic VK*Myc myeloma cells and anti-mouse BCMA CAR T cells. Additionally, the combinatorial effects of opaganib and 4-1BB agonist were examined in VK* Myc myeloma mouse model. Results: When we injected VK*Myc myeloma cells IV into SK1 -/- KO, SK2 -/- KO or WT mice, none of SK2 -/- KO mice developed myeloma or died from myeloma whereas 85-90% of WT recipient mice and all SK1 -/- KO mice developed myeloma and died. Similarly, tumor development was significantly attenuated in SK2 -/- KO mice when CT-2A gliomas cells (intracranial injection), B16F10 melanoma cells (IV) or TRAMP C2 prostate cancer cells (IV) were implanted, demonstrating broad anti-tumor effects of SK2 deletion. We found that the homing of myeloma cells was not affected in SK2 -/- KO mice. SK2 -/- KO recipient mice showed significantly increased number of CD8 T cells and decreased number of MDSCs. Depletion of CD8 T cells using CD8 antibody rendered SK2 -/- KO mice susceptible to myeloma development and administration of anti-Gr-1 antibody increased CD8 T cells and made SK2 WT mice resistant to myeloma development. Adaptive transfer of SK2 -/- KO CD8 T cells suppressed myeloma development, indicative of a critical role of SK2 in T cell regulation. Cytokine profiling revealed significantly increased level of IFNγ and IL-12 and suppressed level of IL-6, GM-CSF, CCL2, TNFα, and IL-10 in SK2 -/- KO recipient mice. CD8 T cells isolated from SK2 -/- KO mice were more proliferative in response to CD3/CD28 antibody stimulation, had higher expression of CD69, Granzyme B and IFNγ but lower level of PD-1, LAG-3, TIGIT and TIM-3, and were more cytotoxic against myeloma cells. Differential gene expression analyses showed up-regulation of IL-36gamma and kallikrein 1-related peptidase b22 (KLK1B22) in SK2 -/- KO CD8 T cells. Knockout of IL-36gamma or KLK1B22 with CRISPR/Cas9 reversed the effects of SK2 deletion on T cell activation and exhaustion, demonstrating the important role of IL-36gamma and KLK1B22 in SK2 mediated T cell regulation. Compared to T cells from WT mice, T cells from SK2 -/- KO mice exhibited enhanced anti-myeloma activities in our VK*Myc myeloma anti-BCMA CAR T mouse model. Finally, treatment of mice with opaganib increased the number of T cells and decreased the number of MDSCs and showed synergistic anti-myeloma activities when combined with 4-1BB agonist in VK*Myc myeloma model. Conclusions: Our study demonstrated that inhibition of SK2 enhances anti-tumor immunity by promoting CD8 T cell activation likely through IL-36gamma and KLK1B22. These studies provide rationale for clinical trials investigating the combination of opaganib with CAR T therapy or other immunotherapy in cancer treatment.

Targeting BCL-XL with a Novel VHL-Based BCL-XL Degrader DT-2216 in Pre-Clinical JAK2-Mutant AML Post-MPN Models

INTRODUCTION Outcomes of acute Myeloid Leukemia (AML) progressed from JAK2-mutant ( JAK2-mut) myeloproliferative neoplasms (MPNs) (post-MPN AML) are dismal, with limited responsiveness to cytotoxic therapies, hypomethylating agents, BCL-2 inhibitor or JAK2 inhibitor, with a median overall survival of less than 1 year ( Dunbar, et al. Blood, 2020). BCL-xL has been found to be overexpressed in cells from JAK2-mut MPN patients ( Petiti et al. J Cell Mol Med, 2020), thus making it a potential therapeutic target. However, the clinical utility of navitoclax, a BCL-xL and BCL-2 dual inhibitor, as demonstrated in combination with JAK2 inhibitor ruxolitinib in patients with myelofibrosis ( Harrison et al. J Clin Oncol, 2022), has been notable for an on-target thrombocytopenia due to the essential role of BCL-xL in platelet survival. In contrast, DT2216, a VHL-recruiting BCL-xL PROTAC, exhibits potent degradation of BCL-xL in leukemia cells while sparing platelets due to minimal expression of VHL in platelets ( Khan et.al Nat. Med, 2019). Consequently, DT2216 may represent an attractive strategy to target BCL-xL, reducing the apoptotic threshold and increasing responsiveness to chemotherapy selectively in tumor cells. In this study, we evaluated the pre-clinical efficacy of DT2216 in combination with ruxolitinib, 5-azacytidine (AZA), or MCL-1 inhibitor S63845 in JAK2-mut AML models. METHODS To validate the dependency of BCL2L1 (gene encoding BCL-xL) on JAK2-mut AML, we analyzed genome-wide CRISPR screen data, RNA-seq data from the DepMap portal and Beat AML 1.0 cohort. To assess the efficacy of DT2216-based treatments, including DT2216 alone, DT2216/AZA combination, DT2216/ruxolitinib combination, and DT2216/S63845 combination, we conducted the Cell Titer Glo (CTG) assay using JAK2-mut AML cell lines and primary leukemia samples. Since most JAK2-mut AML patients are resistant to ruxolitinib, we included ruxolitinib-resistant (ruxo-re) cell lines. The combination index (CI) values obtained at the ED 50, ED 75, and ED 90 were used to determine synergism (CI <1) between the agents, with lower values indicating stronger synergism. Clonogenic assays with three distinct JAK2-mut AML patient samples were performed using DT2216 and/or AZA to assess the anti-leukemia effects on hematopoietic stem and progenitor cells (HSPCs). Western blot was used to determine BCL-xL degradation and measure the expression of BCL-2 and MCL-1 after drug treatments. RESULTS The analysis of BCL2L1 gene expression (p<0.05, JAK2-mut AML cell lines versus other AML cell lines; p<0.01, post-MPN AML versus de novo AML) and BCL2L1 CRISPR gene effect (p<0.05, JAK2-mut AML cell lines versus other AML cell lines) confirmed the role of BCL-xL as a target in JAK2-mut AML. DT2216 as a single agent significantly reduced cell viability in JAK2-mut AML cell lines, with an average IC 50 of 1.61 μM in parental cell lines (range 0.74 - 2.35 μM) and an average IC 50 of 5.37 μM in ruxo-re cell lines (range 1.29 - 8.99 μM) at 72 hours. Notably, DT2216 also markedly reduced cell viability in primary samples (n=3), with an average IC 50 value of 1.21 μM (range 0.41 - 2.59 μM) at 24 hours. Moreover, the average CIs of DT2216/S68425 combination, DT2216/AZA and DT2216/ruxolitinib combination were 0.13 (range 0.07 - 0.17), 0.17 (range 0.61 - 0.22), 0.36 (range 0.14 - 0.54), respectively at 72 hours. Clonogenic assays in three primary JAK2-mut AML samples demonstrated a 69% (range 58.3 - 77.5%) reduction of colony count when treated with DT2216 alone (50 nM) (p<0.001, versus DMSO group) and 76% (range 70.8 - 79.8%) reduction when treated with the combination of DT2216 (50 nM) with AZA (100 nM) (p<0.0001, versus DMSO group) (Fig. 1A). Western blot analysis demonstrated efficient degradation of the BCL-xL protein, with average reductions of 78.5% (range: 70 - 90%), 82.75% (range: 68 - 96%) (Fig. 1B), and 86.75% (range: 83 - 97%) after 24 hours of treatment with DT2216 alone or in combination with AZA or ruxolitinib in both the parental and ruxo-re SET2 and HEL cell lines. CONCLUSIONS These findings highlight the promising efficacy of DT2216 in JAK2-mut AML, as evidenced by reduced cell viability, on-target degradation of BCL-xL, and synergistic anti-leukemia effects when combined with AZA, ruxolitinib or MCL-1 inhibitor. These results provide valuable insights into future therapeutic strategies for the treatment of JAK2-mut AML, particularly in the context of post-MPN AML.

Venetoclax Has Potent Efficacy in NPM1 mutated AML with Acquired Resistance Associated with Either Perturbed Pro-Survival Signalling or NPM1 wild-Type Populations

Background We have previously reported outcomes for patients (pts) receiving venetoclax (VEN) combined with intensive chemotherapy in fit older pts ≥65 years with newly diagnosed AML (Chua et al, JCO 2020). In contrast to younger populations, NPM1(mut) does confer favorable prognosis in older AML populations. Our prior studies indicate that VEN-based therapies in older pts have promising efficacy in NPM1(mut) AML (DiNardo et al, Blood 2020). To characterise this further, we performed a posthoc analysis of the efficacy of single agent VEN and in combination with chemotherapy in NPM1(mut) AML in the CAVEAT study and explored mechanisms of treatment failure using a multi-omic approach. Methods NPM1 MRD was performed using RT-qPCR (sensitivity 10 -6). Integrated analysis from bulk sequencing (targeted 42-gene NGS, whole exome/genome sequencing (WES/WGS), RNAseq), single cell (sc) proteogenomic and transcriptomic assessments (scDNA plus surface protein, Tapestri MissioBio; scRNAseq, 10x Genomics) were performed. Flow cytometry included analysis of BCL-2, BCL-XL, MCL1 and BFL1 expression. Results Of 85 pts enrolled on the CAVEAT study, 20 had NPM1(mut). Median age of NPM1(mut) vs (wt) was 71 vs 70 years. At a median follow-up of 38.6 months, CR/CRi rates and median OS for NPM1(mut) vs (wt) was 85% vs 72% (p=0.37), and 43.9 vs 15.1 months (p=0.04), respectively. Pts received a 7-day VEN monotherapy pre-phase prior to CAVEAT induction with paired bone marrow (BM) assessments pre- and at day 8 to evaluate VEN sensitivity. NPM1(mut) pts had a greater median relative blast reduction after 7-day VEN at -66% (range -7 to -98%), compared to -37% (range +51 to -94%) for NPM1(wt) (p=0.03). 18/20 NPM1(mut) sub-group pts completed induction and 17 had serial NPM1 MRD assessments. The NPM1(mut) level fell a median of 4.55-log 10 following induction, with 6 (35%) pts achieving MRD negative (MRDneg) status. After all consolidation, 12/16 (75%) were MRDneg. 12/12 (100%) pts in long-term follow up remain MRDneg. We investigated the mechanisms of resistance in the 5/20 pts with NPM1(mut) whose disease was primary refractory (RD; n=1) or had acquired secondary resistance (Fig). Two patients (CAL-045, CAL-021) had shown BM blast reductions of ≥45% during VEN pre-phase. Subsequently, at the time of clinical treatment failure, both were NPM1(mut) MRD negative indicating eradication of the starting NPM1(mut) clone and early evolution of a new NPM1(wt) population as the mechanism of resistance. Detailed genomic analysis of CAL-045 showed that a TET2/CBL/NPM1(mut) clone was eradicated, with persistence of TET2 and TET2/CBL(mut) clones lacking NPM1(mut) at the time of refractory disease. scRNAseq analysis of these NPM1(wt) clones revealed a pro-inflammatory signature with elevation in BFL1, a pro-survival gene not targeted by VEN (Fig). In patient CAL-021, NPM1(mut) was also absent with the emergence of nonsense/frameshift BAX variants at relapse as previously described (Moujalled et al, Blood 2022). BAX deficiency is associated with resistance to BH3-mimetics. Two other cases (CAL-014, CAL-020) showed BM blast reductions of <45% during VEN pre-phase. In CAL-014, the expansion of an NPM1 subclone was associated with FLT3-ITD. WES revealed evolution of FLT3-ITD loss of heterozygosity at relapse. Flow cytometric analysis demonstrated disproportionate upregulation of intracellular MCL-1 (Fig). In case CAL-020, no obvious genetic determinants of resistance were identified at relapse, with persistence of a DNMT3A/ KIT/ NPM1(mut) clone (VAF~50%). Whole transcriptome analysis revealed skewed expression of a KIT(mut)allele (from 45% to 88%) at relapse, and protein assessment by flow cytometry revealed marked upregulation of BCL-XL (Fig). Conclusion VEN in combination with intensive chemotherapy for NPM1(mut) AML pts ≥65 years resulted in rapid and deep responses ( NPM1(mut) MRD-negativity) that are associated with prolonged remissions and OS. Mechanisms of treatment failure among pts with NPM1(mut) are diverse and may include kinase-linked upregulation of alternative BCL-2 family pro-survival members or disruption of downstream pro-apoptotic BAX.

Novel Agents with Efficacy Against Cellular Models of Familial Platelet Disorder with Myeloid Malignancy (FPD-MM) Associated with Germline Mutant RUNX1

RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. In addition to mutant (mt) RUNX1, FPD-MM cells commonly harbor co-mutations involving the second allele of RUNX1, or BCOR, PHF6, K-RAS, WT1 or TET2. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need to develop novel targeted therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, conducted with the RNA-Seq signature induced by lethal RUNX1 knockdown in AML cells with mutant (mt) RUNX1, was reported by us to reveal homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole) as the top expression mimickers (EMs). In present studies, we demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in six patient-derived (PD) samples of FPD-MM vs RUNX1-FPD or normal CD34+ progenitor cells. ATAC-Seq and H3K27Ac ChIP-Seq analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater ATAC-Seq peaks and chromatin accessibility in FPD-MM cells with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the population of HSCs but concomitant increase in the macrophage population in FPD-MM (Fig. 1A). This was associated with positive enrichment of the mRNA expression belonging to the gene-sets (HALLMARK) of apoptosis signaling and TP53 targets while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample from a patient with Runx1 K194N, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. CRISPR knockout (KO) RUNX1 led to growth inhibition, such that GMR-AML1 cells that retain Runx1 outcompeted Runx1 KO cells within 20 days. Treatment with HHT induced loss of viability in GMR-AML1 cells (LD50: 40 nM). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation, DNA POLR2B, RRM2, CCND2 and PTPN11. Treatment with MB also dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 330 nM) as well as inhibited cell cycle and protein translation (via OPP Assay). CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the tail-vein infused and engrafted, luciferized GMR-AML1 cell xenograft, compared to the vehicle control, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of the NSG mice (p < 0.05) (Fig. 1B). These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM, including the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are effective against cellular models of FPD-MM versus RUNX1-FPD.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

E7820, an Anti-Cancer Sulfonamide, in Combination with Venetoclax in Patients with Splicing Factor Mutant Myeloid Malignancies: A Phase II Clinical Trial

*JPB and NSC contributed equally. Background and Significance: Mutations in RNA splicing factor genes ( SF3B1, SRSF2 U2AF1 and ZRSR2)are common in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We and others have previously shown that pharmacologic degradation of the RNA splicing factor RBM39 preferentially kills splicing factor mutant cells. These preclinical data provided the rationale for a recently completed phase II clinical trial (NCT05024994) of the oral RBM39 degrader E7820 in patients with relapsed or refractory splicing factor mutant MDS or AML. The safety and efficacy data of this trial have been presented previously (Bewersdorf et al ASH 2022). The generally favorable safety profile and correlative studies confirming target engagement and induction of mis-splicing events support the further exploration of E7820 in the context of combination therapies. Recent clinical data show that treatment with venetoclax can overcome the negative prognostic impact of splicing factor mutations in AML (Senapati et al, Blood 2023). Additionally, preclinical data from our laboratory show synergy between E7820 and venetoclax in SF3B1-mutant isogenic cell lines leading to enhanced RBM39 degradation and blunting of MCL-1 expression, a common cause of venetoclax resistance ( Figure 1). Dynamic BH3 profiling of E7820 in myeloid leukemia cell lines suggests an E7820 dose-dependent priming for BAD which implies forced reliance on BCL2. Furthermore, adding venetoclax to E7820 further increased mitochondrial priming. Preclinical animal modeling with patient-derived xenografts (PDX) is ongoing. Based on these preclinical data, we are amending the study to include a separate arm of E7820 in combination with venetoclax. Methods and Study Design: This is an investigator-initiated, phase II trial (NCT05024994) that is being conducted at Memorial Sloan Kettering Cancer Center and Sylvester Comprehensive Cancer Center at the University of Miami of adult (≥18 years) patients with relapsed or refractory AML, MDS, or chronic myelomonocytic leukemia (CMML). Patients must have a hotspot splicing factor mutation in SF3B1, SRSF2, U2AF1 or U2AF2 (with hotspot mutations as defined by OncoKB) or a nonsense or frameshift mutation in ZRSR2 detected within 6 months of study screening for enrollment. Patients are required to have adequate end-organ function and an Eastern Cooperative Oncology Group (ECOG) performance status of ≤3. Prior treatment with venetoclax is permitted. Patients enrolled in the new combination arm will receive E7820 100mg by mouth once daily + venetoclax orally once daily on days 1-28 of cycle 1 on days 1-21 of subsequent cycles. The study design is shown in Figure 2. As this combination has never been studied in human AML and MDS patients previously, the study will include a 6-patient safety run-in phase with an optional dose de-escalation of E7820 to 70mg orally once daily. The sample size is based on a Minimax Simon 2-stage design. Assuming a type I error rate of 0.05, power of 0.8, a response rate of 10% in the historical control group and 30% with E7820 + venetoclax, 15 patients will be enrolled in stage 1. If there are at least 2 responses among the first 15 patients, we will enroll another 10 patients. The null hypothesis will be rejected if there are at least 6 responses in the 25 patients. The primary endpoint of this trial is to evaluate the efficacy of E7820 + venetoclax in patients with relapsed/refractory myeloid malignancies with mutations in splicing factor genes as measured by the response rate within 6 cycles of therapy (AML: complete remission (CR) + complete remission with partial hematologic recovery (CRh); MDS: CR + CRh + CR with limited hematologic recovery (CR-L) + partial remission (PR); CMML: CR + PR). Secondary endpoints include overall and event-free survival. Correlative biomarker and pharmacodynamic parameters will be assessed as exploratory endpoints including effects on RBM39 protein levels, changes in global and key target splicing events, and evaluation of DCAF15 mRNA levels and response to therapy.

ABC Regimen (Azacitidine, Venetoclax and Chidamide) Is Comparable to the “3+7” Regimen in Newly Diagnosed AML: A Real-World Experience from South China

Background: Traditional “3+7” intense chemotherapy (IC) regimen is the standard treatment backbone for fitness patients with newly diagnosed AML. The azacitidine plus venetoclax (AB) regimen has recently been recommended for elderly AML patients or those ineligible for IC treatment. However, neither the “3+7” IC nor “AB” regimen unmet the needs ofadequate safety and effectiveness, especially on aspects of early death and some subtypes of leukemia, such as M5, or with RUNX1, FLT3-ITD, TP53 mutation, or with MCL-1 up-expression. Chidamide, a novel oral histone deacetylase inhibitor, can overcome the upregulating of MCL-1 expression induced by venetoclax, and synergy with azacitidine and venetoclax induces apoptosis of acute myeloid leukemia cells. Azacitidine-Venetoclax -Chidamide (ABC) combination regimen had been successfully used for refractory relapsed AML and newly diagnosed AML-M5 by Prof. Bin Xu and Prof. Shengli Xue, respectively. Whether the ABC regimen can be comparable to the “3+7” IC regimen in newly diagnosed AML induction therapy is still unknown. Aim: This pilot study aims to evaluate the efficacy and safety of the ABC regimen versus the “3+7” IC regimen in newly diagnosed AML. Method: A total of 112 patients with newly diagnosed AML (not APL) were enrolled in this study. Thirty-one patients from the ABC Collaboration Group were unfitness or unwilling to undergo IC therapy and received ABC regimen treatment from May 2022 to July 2023. Eighty-one patients from GDPH in the AML Collaboration Group of South China were treated with a “3+7” (IC) regimen between November 2010 and August 2013. Treatment protocol: The ABC-28d protocol consists of azacitidine 75mg/M 2, d1-7; venetoclax 100mg d1, 200mg d2, 400mg d3-28, and Chidamide 10mg, d1-6/week for 2 weeks, 28 days as one cycle. The ABC-14d regimen consists of azacitidine 75mg/M 2, d1-7; venetoclax 100mg d1, 200mg d2, 400mg d3-14, and Chidamide 5mg, d1-6/week for 2 weeks, 28 days as one cycle. “3+7” IC regimen consists of idarubicin 10mg/M 2, d1-3; cytarabine 100 mg/M 2, d1-7. Baseline characteristics for patients are listed in Table 1. We compared the complete response (CR) and overall response rate (ORR= CR+ CR with incomplete recovery of blood counts CRi + partial response PR) after the first course of treatment, cumulative response rate, and adverse events between these two cohorts. Results: The outcome data of the ABC regimen was updated as of July 2023 for a median follow-up of 78 days (range 28- 402) for the ABC cohort. The ORR and CR/CRi rate of the ABC cohort after the first cycle therapy was not significantly different from the IC cohort (80.6% vs 75.3%, P =0.108 and 71% vs 65.8%, P=0.552). And the cumulative response rate of the ABC cohort was 87.1%. In primary AML, the CR rate of patients in the ABC cohort was higher than the IC cohort (84% vs 65.8%, P=0.075). Of concern, all 12 patients with M4/M5 treated with the ABC regimen achieved CR (100% vs 76.2%, P=0.03). In secondary AML, the ABC regimen was also poor (CR/CRi rate 33.3%). The ABC regimen is not only applicable to the elder patients (CR rate 100%, 15/15) but also young patients(<60 Y) same as the IC regimen ( CR rate: 71.4% vs 65.8%, P=0.726). The response was summarized in Table 1 and Figure 1. Up to 30 July 2023, the ABC cohort has not reached the median remission duration and overall survival (OS). Grade 3 or 4 hematologic adverse events occurred in 81.2% of patients. Neutrophil and platelet median recovery times were 21 days(0-58) and 16 days (0-58), respectively. Patients in the ABC-14d cohort had lower rates of hematologic AEs than ABC-28d cohort (Neutrophil: 12.13±14.33 vs 26.33±16.89, P =0.024 and Platelet: 11.88±13.92 vs 24.58±18.59, P = 0.048). The most frequent non-hematologic AEs of any grade were neutropenic fever (n=15), pneumonia (n=8) and reversible nephrotoxicity (n=4). In the ABC cohort, no patient experienced early death at week 4 or 8. Conclusions: This preliminary study showed that the safety and efficacy of the ABC regimen were comparable to the traditional “3+7” regimen for newly diagnosed AML, even better for the subtype of AML-M4/M5. The ABC-14 days regimen showed comparable efficacy but better tolerability. However, whether the ABC regimen can replace the traditional intense chemotherapy needs to be confirmed by further randomized controlled trials.

AML under the Scope: Current Strategies and Treatment Involving FLT3 Inhibitors and Venetoclax-Based Regimens.

Acute myeloid leukemia (AML) is a disease that mainly affects elderly patients who are more often unfit for intensive chemotherapy (median age of diagnosis is 68). The regimens, including venetoclax, a highly specific BCL-2 (B-cell lymphoma-2) inhibitor, are a common alternative because of their safer profile and fewer side effects. However, the resistance phenomenon of leukemic cells necessitates the search for drugs that would help to overcome the resistance and improve treatment outcomes. One of the resistance mechanisms takes place through the upregulation of MCL-1 and BCL-XL, preventing BAX/BAK-driven MOMP (mitochondrial outer membrane permeabilization), thus stopping the apoptosis process. Possible partners for BCL-2 inhibitors may include inhibitors from the FLT3i (FMS-like tyrosine kinase-3 inhibitor) group. They resensitize cancer cells through the downregulation of MCL-1 expression in the FLT3 mutated cells, resulting in the stronger efficacy of BCL-2 inhibitors. Also, they provide an additional pathway for targeting the clonal cell. Both preclinical and clinical data suggest that the combination might show a synergistic effect and improve patients' outcomes. The aim of this review is to determine whether the combination of venetoclax and FLT3 inhibitors can impact the therapeutic approaches and what other agents they can be combined with.

Combination of MCL-1 and BCL-2 inhibitors is a promising approach for a host-directed therapy for tuberculosis

Tuberculosis (TB) accounts for 1.6 million deaths annually and over 25% of deaths due to antimicrobial resistance. Mycobacterium tuberculosis (M.tb) drives MCL-1 expression (family member of anti-apoptotic BCL-2 proteins) to limit apoptosis and grow intracellularly in human macrophages. The feasibility of re-purposing specific MCL-1 and BCL-2 inhibitors to limit M.tb growth, using inhibitors that are in clinical trials and FDA-approved for cancer treatment has not be tested previously. We show that specifically inhibiting MCL-1 and BCL-2 induces apoptosis of M.tb-infected macrophages, and markedly reduces M.tb growth in human and murine macrophages, and in a pre-clinical model of human granulomas. MCL-1 and BCL-2 inhibitors limit growth of drug resistant and susceptible M.tb in macrophages and act in additive fashion with the antibiotics isoniazid and rifampicin. This exciting work uncovers targeting the intrinsic apoptosis pathway as a promising approach for TB host-directed therapy. Since safety and activity studies are underway in cancer clinics for MCL-1 and BCL-2 inhibitors, we expect that re-purposing them for TB treatment should translate more readily and rapidly to the clinic. Thus, the work supports further development of this host-directed therapy approach to augment current TB treatment.

Synthesis by diastereomeric resolution, biochemical evaluation and molecular modelling of chiral 3-hydroxyl b-lactam microtubule-targeting agents for the treatment of triple negative breast and chemoresistant colorectal cancers

The synthesis and biochemical activity of a series of chiral trans 3-hydroxyl β-lactams targeting tubulin is described. Synthesis of the series of enantiopure β-lactams was achieved using chiral derivatising reagent N-Boc-l-proline. The absolute configuration was determined as 3S,4S for (+) enantiomer 4EN1 and 3R,4R for (-) enantiomer 4EN2. Antiproliferative studies identified chiral 3S,4S b-lactams with subnanomolar IC50 values across a range of cancer cell lines, improving potency with respect to the corresponding racemates. Fluoro-substituted (+)-(3S,4S)-4-(3-fluoro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27EN1) was determined as the lead eutomer with dual antiproliferative activity in triple negative breast cancer cells (TNBC), and combretastatin A-4 resistant HT-29 colorectal cancer cells. IC50 values were in the range of 0.26–0.7 nM across four cell lines. Tubulin polymerisation assays, confocal microscopy and molecular modelling studies indicated that 3S,4S eutomers are microtubule destabilisers, while 3R,4R distomers have lower potency as microtubule destabilisers. 27EN1 demonstrated anti-mitotic and pro-apoptotic activity in MDA-MB-231 and HT-29 cells in addition to selective toxicity toward MCF-7 breast cancer versus non-tumorigenic MCF-10-2A cells. The related 3S,4S β-lactam eutomer 4EN1 downregulated expression of key cell survival anti-apoptotic proteins Bcl-2 and Mcl-1 in MDA-MB-231 cells while 27EN1 downregulated Mcl-1 in HT-29 cells. Chiral β-lactam 27EN1 will be further developed for treatment of TNBC and CA-4 resistant colorectal cancers.

MTORC1-selective activation of translation elongation promotes disease progression in chronic lymphocytic leukemia

Targeted deletion of Raptor, a component of mechanistic target of rapamycin complex 1 (mTORC1), reveals an essential role for mTORC1 in initiation/maintenance of leukemia in a CLL model, resulting from a failure for haemopoietic stem/progenitor cells (HSPCs) to commit to the B cell lineage. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor CLL progenitor cells (PKCα-KR-transduced HSPCs) after disease establishment revealed a reduction in CLL-like disease load and a significant increase in survival in the mice. Interestingly in an aggressive CLL-like disease model, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive disease. Rapamycin, but not ibrutinib, efficiently targeted the eEF2/eEF2K translation elongation regulatory axis, downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. mTOR inhibitor treatment of primary patient CLL cells halted proliferation, at least in part through modulation of eEF2K/eEF2 phosphorylation and expression, reduced protein synthesis and inhibited expression of MCL1, Cyclin A and Cyclin D2. Our studies highlight the importance of translation elongation as a driver of disease progression and identify inactivation of eEF2 activity as a novel therapeutic target for blocking CLL progression.

RETRACTED ARTICLE: Upregulation of MCL-1 by LUCAT1 through interacting with SRSF1 promotes the migration and invasion in non-small cell lung carcinoma.

The onset of non-small cell lung carcinoma (NSCLC) still be in the fog. LUCAT1 is potentially capable of modulating MCL-1-involved NSCLC pathogenesis via targeting SRSF1. Also, MCL-1 can regulate Wnt/β-catenin pathway to affect the tumorigenesis of NSCLC. Thus, this paper aims to uncover an intriguing and novel role of LUCAT1/SRSF1/MCL-1 axis in NSCLC based on Wnt/β-catenin pathway. A549 and NCI-H1650, two cell lines of NSCLC, were used to mimic NSCLC in vitro. MCL-1 siRNA (si-MCL-1) and LUCAT1 siRNA (si-LUCAT1) were used to downregulate MCL-1 and LUCAT1 in NSCLC cells, respectively. The overexpression vector of SRSF1 based on pcDNA 3.1 was constructed to upregulate SRSF1 expression. 40 μM SKL2001 was used to activate Wnt/β-catenin pathway. Transwell assay was used for migrative and invasive tests. The effect of LUCAT1 on tumor metastasis was verified in nude mice. MCL-1 downregulation led to the decrease of EMT, invasion, and migration in NSCLC, while Wnt/β-catenin pathway agonist partially reversed the effects of MCL-1 downregulation. Mechanistic investigations revealed that LUCAT1 and MCL-1 mRNA were enriched in SRSF1; LUCAT1 silence decreased MCL-1, whereas SRSF1 enhancement elevated MCL-1; Importantly, SRSF1 overexpression significantly reversed MCL-1 alteration due to LUCAT1 silence. In NSCLC cells, SRSF1 overexpression offset the si-LUCAT1-induced changes, and si-MCL-1 reversed the SRSF1-induced cellular changes. Further, LUCAT1 inhibition reduced lung metastasis of cancer cells. LUCAT1 can interact with SRSF1 to regulate MCL-1 expression that targets the Wnt/β-catenin pathway-mediated NSCLC cell migration, invasion, and EMT.

Increased MCL1 dependency leads to new applications of BH3-mimetics in drug-resistant neuroblastoma

BackgroundNeuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells.MethodsWe utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance.ResultsIn a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells.ConclusionsThese data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.

Soybean lectin-triggered IL-6 secretion induces autophagy to kill intracellular mycobacteria through P2RX7 dependent activation of the JAK2/STAT3/Mcl-1 pathway

Cytokine therapy and cytokine-mediated autophagy have been used as prominent host-directed therapy (HDT) approaches to restrain M. tb growth in the host cell. In the present study, we have dissected the anti-tubercular activity of Soybean lectin (SBL) through cytokine-mediated autophagy induction in differentiated THP-1 (dTHP-1) cells. A significant increase in IL-6 expression was observed in both uninfected and mycobacteria infected dTHP-1 cells through the P2RX7 mediated pathway via PI3K/Akt/CREB-dependent signalling after SBL treatment. Inhibition of IL-6 level using IL-6 neutralizing antibody or associated signalling significantly enhanced the mycobacterial load in SBL-treated dTHP-1 cells. Further, autocrine signalling of IL-6 through its receptor-induced Mcl-1 expression activated autophagy via JAK2/STAT3 pathway, and inhibition of this pathway affected autophagy. Finally, blocking the IL-6-regulated autophagy through NSC 33994 (a JAK2 inhibitor) or S63845 (an Mcl-1 inhibitor) led to a notable increase in intracellular mycobacterial growth in SBL-treated cells. Taken together, these results indicate that SBL interacts with P2RX7 to regulate PI3K/Akt/CREB network to release IL-6 in dTHP-1 cells. The released IL-6, in turn, activates the JAK2/STAT3/Mcl-1 pathway upon interaction with IL-6Rα to modulate autophagy that ultimately controls mycobacterial growth in macrophages.

The outer membrane protein Tp92 of Treponema pallidum delays human neutrophil apoptosis via the ERK, PI3K/Akt, and NF-κB pathways.

Syphilis is a persistent sexually transmitted disease caused by infiltration of the elusive pathogen Treponema pallidum. Despite the prevalence of human polymorphonuclear neutrophils (hPMNs) within cutaneous lesions, which are characteristic of incipient syphilis, their role in T. pallidum infection remains unclear. Tp92 is the only T. pallidum helical outer membrane protein that exhibits structural features similar to those of outer membrane proteins in other gram-negative bacteria. However, the functional mechanism of this protein in immune cells remains unclear. Neutrophils are short-lived cells that undergo innate apoptosis in response to external stimuli that typically influence this process. In this study, we determined that Tp92 impedes the activation of procaspase-3 via the ERK MAPK, PI3K/Akt, and NF-κB signaling pathways, consequently suppressing caspase-3 activity within hPMNs, and thereby preventing hPMNs apoptosis. Furthermore, Tp92 could also modulate hPMNs apoptosis by enhancing the expression of the anti-apoptotic protein Mcl-1, stimulating IL-8 secretion, and preserving the mitochondrial membrane potential. These findings provide valuable insights into the molecular mechanisms underlying T. pallidum infection and suggest potential therapeutic targets for syphilis treatment.

Cordycepin improves sensitivity to temozolomide in glioblastoma cells by down-regulating MYC.

Glioblastoma is one of the malignant tumors with poor prognosis and no effective treatment is available at present. To study the effect of cordycepin combined with temozolomide on glioblastoma, we explored the effect of the combination based on network pharmacology and biological verification. It was found that the drug combination significantly inhibited the cell growth, proliferation, migration and invasion of LN-229 cells. Drug combination inhibited epithelial-mesenchymal transition (EMT) by up-regulating the expression of E-cadherin and suppressing the expression of N-cadherin, Zeb1 and Twist1. Through network pharmacology, we further explored the molecular mechanism of drug combination against glioblastoma, and 36 drug-disease common targets were screened. The GO biological process analysis included 44 items (P < 0.01), which mainly involved the regulation of apoptosis, cell proliferation, cell migration, etc. The enrichment analysis of KEGG pathways included 28 pathways (P < 0.05), and the first four pathways were "MicroRNA in cancer, Proteoglycans in cancer, Pathways in cancer and PI3K-AKT signaling pathway". We detected the expression of important genes in the pathways and PPI network, and the results showed that the drug combination down-regulated NFKB1, MYC, MMP-9, MCL1, CTNNB1, and up-regulated PDCD4. Cordycepin combined with temozolomide may down-regulate MYC through "MicroRNA in cancer, Proteoglycans in cancer, Pathways in cancer and PI3K-AKT signaling pathway", which in turn regulate the expression of MCL1, CTNNB1, MMP9, PDCD4, thus regulating cell proliferation, migration and apoptosis in glioblastoma.

Host-derived growth factors drive ERK phosphorylation and MCL1 expression to promote osteosarcoma cell survival during metastatic lung colonization.

For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.