Downregulation Of Mcl-1 Research Articles

Integrated stress response activation induced by usnic acid alleviates BCL-2 inhibitor ABT-199 resistance in acute myeloid leukemia

IntroductionABT-199 (venetoclax) is a BCL-2 suppressor with pronounced effects on acute myeloid leukemia (AML). However, its usefulness as a monotherapy or in combination with hypomethylating medicines like azacitidine is debatable due to acquired resistance. Usnic acid, a dibenzofuran extracted from lichen Usnea diffracta Vain, exhibits anticancer properties and may counteract multidrug resistance in leukemia cells. ObjectiveThis study investigated whether usnic acid at low-cytotoxicity level could enhance sensitivity of AML cells with acquired resistance to ABT-199 by targeting the integrated stress response pathways. MethodsTo investigate the combined effects on AML cells, we used a cell viability test, flow cytometry to quantify apoptosis, cell cycle analysis, and mitochondrial membrane potential measurement. RNA-seq and immunoblot were used to determine the potential mechanisms of ABT-199 + usnic acid combination. ResultsUsnic acid, at a low cytotoxicity level, successfully restored ABT-199 sensitivity in AML cell lines that had developed ABT-199 resistance and increased ABT-199′s antileukemic activity in a xenograft model. Mechanistically, the combination of usnic acid and ABT-199 cooperated to boost the expression of the integrated stress response (ISR)-associated genes ATF4, CHOP, and NOXA through the heme-regulated inhibitor kinase (HRI), while also promoting the degradation of the anti-apoptotic protein MCL-1. ISRIB, a compound that blocks the ISR, was able to reverse the growth suppression and cell death, the increase in expression of genes related with the ISR, and the inhibition of MCL-1 protein caused by combination therapy. Additionally, the downregulation of MCL-1 was linked to an increase in MCL-1 phosphorylation at serine 159 and subsequent destruction by the proteasome. ConclusionIn summary, usnic acid improves chemosensitivity to ABT-199 by triggering the integrated stress response, leading to decreased levels of MCL-1 protein, suggesting a potential treatment for AML cases resistant to Bcl-2 inhibitors.

Wogonin induces mitochondrial apoptosis and synergizes with venetoclax in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is among the most aggressive hematological malignancies and patients are commonly treated with combinatorial immunochemotherapies such as R-CHOP. Till now, the prognoses are still variable and unsatisfactory, depending on the molecular subtype and the treatment response. Developing effective and tolerable new agents is always urgently needed, and compounds from a natural source have gained increasing attentions. Wogonin is an active flavonoid extracted from the traditional Chinese herbal medicine Scutellaria baicalensis Georgi and has shown extensive antitumor potentials. However, the therapeutic effect of wogonin on DLBCL remains unknown. Here, we found that treatment with wogonin dose- and time-dependently reduced the viability in a panel of established DLBCL cell lines. The cytotoxicity of wogonin was mediated through apoptosis induction, along with the loss of mitochondrial membrane potential and the downregulation of BCL-2, MCL-1, and BCL-xL. In terms of the mechanism, wogonin inhibited the PI3K and MAPK pathways, as evidenced by the clear decline in the phosphorylation of AKT, GSK3β, S6, ERK, and P38. Furthermore, the combination of wogonin and the BCL-2 inhibitor venetoclax elicited synergistically enhanced killing effect on DLBCL cells regardless of their molecular subtypes. Finally, administration of wogonin significantly impeded the progression of the DLBCL tumor in a xenograft animal model without obvious side effects. Taken together, the present study suggests a promising potential of wogonin in the treatment of DLBCL patients either as monotherapy or an adjuvant for venetoclax-based combinations.

Marine Cytotoxin Santacruzamate A Derivatives as Potent HDAC1-3 Inhibitors and Their Synergistic Anti-Leukemia Effects with Venetoclax.

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by infiltration of the blood and bone marrow, exhibiting a low remission rate and high recurrence rate. Current research has demonstrated that class I HDAC inhibitors can downregulate anti-apoptotic proteins, leading to apoptosis of AML cells. In the present investigation, we conducted structural modifications of marine cytotoxin Santacruzamate A (SCA), a compound known for its inhibitory activity towards HDACs, resulting in the development of a novel series of potent class I HDACs hydrazide inhibitors. Representative hydrazide-based compound 25c exhibited concentration-dependent induction of apoptosis in AML cells as a single agent. Moreover, 25c exhibited a synergistic anti-AML effect when combined with Venetoclax, a clinical Bcl-2 inhibitor employed in AML therapy. This combination resulted in a more pronounced downregulation of anti-apoptotic proteins Mcl-1 and Bcl-xL, along with a significant upregulation of the pro-apoptotic protein cleaved-caspase3 and the DNA double-strand break biomarker γ-H2AX compared to monotherapy. These results highlighted the potential of 25c as a promising lead compound for AML treatment, particularly when used in combination with Venetoclax.

ONC212 enhances YM155 cytotoxicity by triggering SLC35F2 expression and NOXA-dependent MCL1 degradation in acute myeloid leukemia cells

Although the anticancer activity of ONC212 has been reported, the precise mechanism underlying its apoptotic effects remains unclear. In this study, we investigated the apoptotic mechanism of ONC212 in acute myeloid leukemia (AML) cells. ONC212 induces apoptosis, MCL1 downregulation, and mitochondrial depolarization in AML U937 cells. Ectopic MCL1 expression alleviates mitochondria-mediated apoptosis in ONC212-treated U937 cells. ONC212 triggers AKT phosphorylation, inducing NOX4-dependent ROS production and promoting HuR transcription. HuR-mediated ATF4 mRNA stabilization stimulates NOXA and SLC35F2 expression; ONC212-induced upregulation of NOXA leads to MCL1 degradation. The synergistic effect of ONC212 on YM155 cytotoxicity was dependent on increased SLC35F2 expression. In addition, YM155 feedback facilitated the activation of the ONC212-induced signaling pathway. A similar mechanism explains ONC212- and ONC212/YM155-induced AML HL-60 cell death. The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212. In U937/HQ cells, ONC212 triggered apoptosis through NOXA-mediated MCL1 downregulation, enhancing YM155 cytotoxicity. Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity.

Abstract 1966: The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia

Abstract In approximately 23% of AML patients, Fms-like tyrosine kinase 3 (FLT3) contains a gain-of-function, internal tandem duplication (ITD) mutation that is associated with an unfavourable prognosis. FLT3 is therefore seen as a promising therapeutic avenue for AML and as a result, a more complete understanding of its function and signalling may lead to additional targets and resistance mechanisms being identified. To date, several FLT3 inhibitors including Quizartinib (AC220), a second-generation ITD-selective tyrosine kinase inhibitor, have been developed, but single-agent clinical trials have not been overwhelmingly successful. In most preclinical studies, the inhibitory effects of FLT3 inhibitors are mainly evaluated using mutant-expressing models, however, most AML patients harbour both a wild-type and mutant FLT3 allele as well as presenting with high plasma levels of FLT3 ligand (FLT3L). We hypothesized that FLT3L could act through WT-FLT3 to influence the efficacy of FLT3 inhibitors in cells with heterozygous mutations. In this study, we have examined the role of FLT3 inhibition and FLT3L activation on the cellular localisation and downstream signalling in several AML cell lines, some of which express ITD mutations. We also looked at mechanisms by which FLT3L could impair the efficacy of FLT3 inhibitors. Our results revealed that the majority of FLT3 in the MV4-11 and MOLM-13 cells was intracellular. Inhibition of ITD-FLT3 with quizartinib led to a dramatic relocalisation of FLT3 to the cell surface. This effect was more pronounced in MV4-11 (FLT3ITD/ITD) than the heterozygous MOLM-13 (FLT3ITD/WT) cells that also express WT. This was accompanied by inhibition of ERK, AKT and STAT5 signalling pathways and resulted in cell death. Quizartinib induced cell death only in AML lines expressing FLT3-ITD mutations (MOLM-13 and MV4-11), and this could be antagonised by FLT3 ligand (FLT3L). The largest inhibitory effects were seen in heterozygous cells expressing a mutated and wild-type allele. FLT3 inhibition was associated with downregulation of the anti-apoptotic protein Mcl1 and upregulation of the pro-apoptotic BH3-only protein, Bim. Our experiments indicated that both of these proteins were regulated by both WT and ITD-FLT3 through the MAPK pathway. These results suggest that activation of FLT3 signalling by FLT3L confers resistance to quizartinib through upregulation of Mcl-1 and suppression of Bim expression. Taken together, our data suggest that FLT3 cell surface localisation and expression are controlled by ITD mutations and are key components of drug resistance. The data also suggests a novel therapeutic approach in patients with high plasma FLT3L levels, especially when using type II inhibitors such as quizartinib, is to co-target the MAPK/ERK pathway to abrogate the FLT3Lmediated resistance in FLT3-ITD AML. Citation Format: Taha Alqahtani, David MacEwan. The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1966.

Pim Kinase Inhibitors Increase Gilteritinib Cytotoxicity in FLT3-ITD Acute Myeloid Leukemia Through GSK-3β Activation and c-Myc and Mcl-1 Proteasomal Degradation.

FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3βactivation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.

Vitamin K2 sensitizes the efficacy of venetoclax in acute myeloid leukemia by targeting the NOXA-MCL-1 pathway.

Promising outcomes have been reported in elder patients with acute myeloid leukemia (AML) using combined therapy of venetoclax (VEN) and azacytidine (AZA) in recent years. However, approximately one-third of patients appear to be refractory to this therapy. Vitamin K2 (VK2) shows apoptosis-inducing activity in AML cells, and daily oral VK2 (menaquinone-4, GlakayR) has been approved for patients with osteoporosis in Japan. We observed a high response rate to AZA plus VEN therapy, with no 8-week mortality in the newly diagnosed AML patients consuming daily VK2 in our hospital. The median age of the patients was 75.9 years (range 66-84) with high-risk features. Patients received AZA 75 mg/m2 on D1-7, VEN 400 mg on D1-28, and daily VK2 45 mg. The CR/CRi ratio was 94.7% (18/19), with a CR rate of 79%. Complete cytogenetic CR was achieved in 15 of 19 (79%) patients, and MRD negativity in 2 of 15 (13%) evaluable CR patients. Owing to the extremely high response rate in clinical settings, we further attempted to investigate the underlying mechanisms. The combination of VK2 and VEN synergistically induced apoptosis in all five AML cell lines tested. VK2, but not VEN, induced mitochondrial reactive oxygen species (ROS), leading to the transcriptional upregulation of NOXA, followed by MCL-1 repression. ROS scavengers repressed VK2 induced-NOXA expression and led to the cancellation of pronounced apoptosis and the downregulation of MCL-1 by VK2 plus VEN. Additionally, knockdown and knockout of NOXA resulted in abrogation of the MCL-1 repression as well as enhanced cytotoxicity by the two-drug combination, indicating that VK2 suppresses MCL-1 via ROS-mediated NOXA induction. These data suggest that the dual inhibition of BCL-2 by VEN and MCL-1 by VK2 is responsible for the remarkable clinical outcomes in our patients. Therefore, large-scale clinical trials are required.

Abstract LB_A05: Coadministration of AZD 4573 and gilteritinib exhibits a potent activity in FLT3 inhibitor-sensitive and -resistant AML cells

Abstract Acute myeloid leukemia (AML) is caused by the sequential acquisition of mutations that regulate multiple cellular pathways, indicating that multiple pathways must be coordinately inhibited for a successful therapy in this disease. Transcriptomics analysis suggest that exposure of AML cells to the selective CDK9 inhibitor AZD4573 led to activation of multiple survival pathways including MAPK, PI3K, and STAT suggesting that disruption of these signaling pathways may potentiate this agent activity. Notably, combined treatment with AZD4573 and the FLT3 inhibitor gilteritinib resulted in a robust suppression of cell growth and a marked induction of apoptosis in various AML cell lines, particularly those with FLT3 mutations. These were associated with a marked induction of apoptosis reflected by increased Annexin V/PI positivity and cleavage in caspase 3 and its substrate PARP. Importantly, combined treatment was also highly effective in FLT3 mutants AML cells that exhibit significant resistance to various FLT3 inhibitors including gilteritinib. Combined treatment led to a profound disruption of critical survival signaling pathways including MEK/ERK, STAT, and PI3K/AKT pathways. Combined treatment also significantly disrupted the balance between the proapoptotic and anti-apoptotic Bcl-2 members as reflected by downregulation of MCL1 and upregulated of Bim protein levels. Importantly, GILT/AZD4573-induced cell death was fully prevented when Bax and Bak were knocked out using CRISPR system, suggesting that the anti-AML activity of this regimen proceeds mainly via the mitochondrial apoptotic pathway. Finally, using a systemic acute myeloid leukemia xenograft model, co-administration of gilteritinib and AZD4573 sharply reduced leukemia growth and markedly prolonged mice survival. Collectively, these findings demonstrate that dual inhibition of CDK9 and FLT3 exhibits a potent anti-leukemic efficacy in vitro and in vivo. These findings also indicate that this strategy warrants further attention in FLT3-dependent acute myeloid leukemias. Citation Format: Mohamed Rahmani, Baraa Khalid Salah, Burcu Yener, Bassam Mahboub, Steven Grant. Coadministration of AZD 4573 and gilteritinib exhibits a potent activity in FLT3 inhibitor-sensitive and -resistant AML cells [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 LB_A05.

Transient CDK9 Inhibition with AZD4573 Effectively Induces Apoptosis in Burkitt Lymphoma As a Monotherapy in Pre-Clinical Models

Burkitt Lymphoma (BL) accounts for almost two thirds of all B-cell non-Hodgkin lymphoma cases in children and adolescents. It may affect the jaw, bones of the face, bowel, kidney, ovaries and in some cases spread to the central nervous system. Nearly all types of BL are characterized by c-MYC gene translocation of chromosome 8 with immunoglobulin genes at chromosome 2, 14 or 22 resulting in high levels of the transcription factor. Recently, MCL-1 was shown to be overexpressed in BL and is a critical survival factor in pre-clinical BL models using genetic and chemical approaches. CDK9, a serine/threonine kinase, regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). AZD4573 is a highly potent and selective CDK9 inhibitor that results in downregulation of short-lived transcripts and labile proteins such as c-MYC and key survival proteins including MCL-1 and BFL-1, resulting in apoptosis in several hematological malignancies (Cidado et al. 2020 & Boiko et al. 2021) We selected several preclinical BL models to assess the rapid apoptogenic potential of AZD4573 in vitro and in vivo. To measure apoptosis, we evaluated cleaved caspase-3 (CC3) induction following acute treatment (6h) using Caspase-Glo 3/7. Using this assay, we found that 3 BL models were sensitive to CDK9 inhibition (EC50 < 100nM; max. CC3 > 50%) and 2 were resistant (EC50 > 100nM; max. CC3 < 50%). Treatment with AZD4573 in these sensitive BL cell line models decreased pSer2-RNAPII, and downregulation of MCL-1 and c-MYC within 6 hours. In AZD4573 resistant lines we observed relatively higher levels of anti-apoptotic proteins BCL2 and BCL-xL, compared to the sensitive cell lines. These increased levels potentially lead to the decreased sensitivity to AZD4573, which does not reduce levels of BCL-2 and BCL-xL due to their longer half-life. Using CRISPR genetic knockouts we demonstrate that BL cell lines are dependent on c-MYC and MCL-1 and that loss of these protein decreases caspase induction after AZD4573 treatment, highlighting the importance of these proteins in driving the efficacy of AZD4573. In vivo AZD4573 dosed on a once weekly schedule resulted in tumor growth inhibition in aggressive BL cell line xenografts Namalwa and Ramos (40-60%) and decreased pSer2-RNAPII, MCL-1 and c-MYC and increased CC3 8 hours after treatment. AZD4573 is currently in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK of AZD4573 in patients with relapsed or refractory Peripheral T-cell lymphomas (pTCL). Our findings demonstrate that targeting CDK9 with AZD4573 can effectively induce apoptosis in pre-clinical BL models and could be an effective therapy for BL patients.

The Novel CDK9/CDK4/6/PI3K Triple Inhibitor LCI139 for the Treatment of MYC-Driven Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy with characteristic t(11;14)(q13;q32) translocation juxtaposing IGH and CCND1 gene loci. This chromosomal rearrangement leads to Cyclin D1 overexpression and cell cycle dysregulation at the G1/S phase transition. MCL is generally diagnosed at advanced stages and follows an aggressive course of disease. Although chemotherapy-based combination regimens are initially effective, most patients relapse with a median survival of only 2.5-5 years. While novel targeted therapeutics have shown promising activity in relapsed/refractory patients, inevitable relapse associated with progressively declining efficacy and increasing resistance to single agent targeted therapy is a major concern. To overcome this barrier, we designed in-silico based a novel multitarget small molecule inhibitor LCI139 which simultaneously blocks three oncogenic targets: cyclin-dependent kinase 9 (CDK9), CDK4/6, and phosphatidylinositol-3 kinase (PI3K). LCI139 is a potent CDK9/CDK4/6/PI3K inhibitor with IC 50s of 0.000039µM (CDK9), 0.0015µM/0.0036µM (CDK4/6) 0.070µM (p110α), 0.461µM (p110γ), and 0.214 (p110δ), as determined by cell free assay alpha screens. Dose-dependent screening of LCI139 against a panel of MCL cell lines revealed robust cytotoxic effects with maximal IC 50 of 86.67 nM in JeKo-1 cells, 64.8 nM in Mino cells, and 202.5 nM in Rec-1 cells. Compared with clinical single agent CDK9 inhibitor AZD4573, LCI139 is less toxic to normal human stromal cells (HS-5), whereas both LCI139 and AZD4573 have no significant cytotoxic effect on normal human foreskin fibroblast cells (Hs68). LCI139 treatment at various concentrations for 24hrs induced significant apoptotic response in two MCL cell lines, JeKo-1 (p<0.001 DMSO vs LCI139 100 nM, p<0.0001 DMSO vs LCI139 200 nM) and Mino (p<0.05 DMSO vs LCI139 100 nM, p<0.0001 DMSO vs LCI139 200 nM) as determined by Annexin V/7-AAD apoptosis staining. Similarly, LCI139-induced apoptotic cell death was further confirmed by increasing cl-PARP protein levels compared to control as assessed by Western blot analysis. LCI139 induced robust G2/M cell cycle arrest in JeKo-1 cells and G1 arrest in Mino cells. The mechanism of action of pan-CDK inhibitors has been associated with downregulation of the anti-apoptotic protein MCL-1 through inhibition of transcriptional CDKs. Our results show that short-term exposure of LCI139 (1hr and 3hr) significantly suppressed MCL-1 and MYC mRNA expression in JeKo-1 (p<0.00001 DMSO vs LCI139) and Mino cells (p<0.00001 DMSO vs LCI139). LCI139 suppressed pRpb1 (Ser2), MCL-1 and cMYC protein expression in IgM-stimulated JeKo-1 and Mino cells in a dose-dependent manner. Treatment with LCI139 had no significant effect on pAKT (Ser473) in Jeko-1 cells, whereas pAKT (Ser473) was completely abolished in Mino cells at 500nM of LCI139. Mechanistically, our findings also demonstrate that LCI139 decreased MCL-1 and cMYC protein stability and reduced half-life as accessed by cycloheximide chase assay. This decrease in cMYC stability is associated with decreased of cMYC phosphorylation at Ser62. Adaptive kinome remodeling via drug-induced transcriptional reprogramming is a known contributor to ibrutinib resistant (IR) MCL. RNA-sequencing analysis of JeKo-1 vs Jeko-1 IR cells revealed a total of 3468 differentially expressed genes (FC > 1.5 and FDR < 0.05) where 110 kinases display altered expression in JeKo-1 IR cells. Finally, we show that LCI139 overcomes chronic ibrutinib resistance by decreasing viability in JeKO-1 IR and Mino IR cells with an IC 50 of 79 nM and 89 nM, respectively. Therefore, the multitarget small molecule inhibitor LCI139 has superior potency against IR MCL cell lines and overcomes ibrutinib-resistance at nanomolar doses.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

Hydroquinone-selected chronic myelogenous leukemia cells are sensitive to chloroquine-induced cytotoxicity via MCL1 suppression and glycolysis inhibition

Previous studies have provided evidence that repeated exposure to the benzene metabolite hydroquinone (HQ) induces malignant transformation and increases basal autophagy in the chronic myeloid leukemia (CML) cell line K562. This study explored the cytotoxicity of the autophagy inhibitor chloroquine (CQ) on parental and HQ-selected K562 (K562/HQ) cells. CQ triggered apoptosis in these cells independently of inhibiting autophagic flux; however, in K562/HQ cells, CQ-induced cytotoxicity was higher than in K562 cells. Mechanistically, CQ-induced NOXA upregulation led to MCL1 downregulation and mitochondrial depolarization in K562/HQ cells. MCL1 overexpression or NOXA silencing attenuated CQ-mediated cytotoxicity in K562/HQ cells. CQ triggered ERK inactivation to increase Sp1, NFκB, and p300 expression, and co-assembly of Sp1, NFκB, and p300 in the miR-29a promoter region coordinately upregulated miR-29a transcription. CQ-induced miR-29a expression destabilized tristetraprolin (TTP) mRNA, which in turn reduced TTP-mediated NOXA mRNA decay, thereby increasing NOXA protein expression. A similar mechanism explained the CQ-induced downregulation of MCL1 in K562 cells. K562/HQ cells relied more on glycolysis for ATP production than K562 cells, whereas inhibition of glycolysis by CQ was greater in K562/HQ cells than in K562 cells. Likewise, CQ-induced MCL1 suppression and glycolysis inhibition resulted in higher cytotoxicity in CML KU812/HQ cells than in KU812 cells. Taken together, our data confirm that CQ inhibits MCL1 expression through the ERK/miR-29a/TTP/NOXA pathway, and that inhibition of glycolysis is positively correlated to higher cytotoxicity of CQ on HQ-selected CML cells.

Blockade of interleukin-6 trans-signaling prevents mitochondrial dysfunction and cellular senescence in retinal endothelial cells

Interleukin-6 (IL-6) is a multifaceted cytokine implicated in the pathogenesis of diabetic retinopathy (DR). Its activity extends through cis- and trans-signaling (TS) pathways, with cis-signaling limited to specific cell types possessing the membrane-bound IL-6 receptor, while trans-signaling broadly activates various cells without the membrane bound IL-6 receptor, including retinal endothelial cells. In this study, we determined the effects of interleukin-6 trans-signaling on mitochondrial dysfunction and cellular senescence in human retinal endothelial cells (HRECs). HRECs were cultured and treated with IL-6 + soluble IL-6R or Hyper IL-6 to activate trans-signaling, along with sgp130Fc for inhibition. RT-PCR was used to analyze gene expression changes associated with inflammation and senescence. Cellular senescence was assessed using SA β-gal staining. Mitochondrial function was evaluated using Seahorse XFe24 Bioanalyzer. IL-6 trans-signaling induced inflammatory gene expression as indicated by the upregulation of ICAM1, MCP1, and SERPINA3 levels. Additionally, it reduced mitochondrial respiration and oxidative phosphorylation, and these effects were counteracted by sgp130Fc. Moreover, IL-6 trans-signaling led to altered expression of apoptosis-associated genes, including downregulation of FIS1, BCL2, and MCL1, while promoting cellular senescence, a phenomenon mitigated by sgp130Fc. These results not only deepen our understanding of IL-6 in DR but also carry broader implications for age-related diseases and the aging process itself. This study underscores the potential therapeutic value of targeting IL-6 trans-signaling with sgp130Fc as a promising anti-inflammatory approach for DR and potentially other inflammatory conditions. Further in-vivo investigations are warranted to elucidate the function of IL-6 trans-signaling in aging-related pathologies and overall organismal health.

BH3 mimetics and azacitidine show synergistic effects on juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive hematopoietic disorder of infancy and early childhood driven by constitutively active RAS signaling and characterized by abnormal proliferation of the granulocytic-monocytic blood cell lineage. Most JMML patients require hematopoietic stem cell transplantation for cure, but the risk of relapse is high for some JMML subtypes. Azacitidine was shown to effectively reduce leukemic burden in a subset of JMML patients. However, variable response rates to azacitidine and the risk of drug resistance highlight the need for novel therapeutic approaches. Since RAS signaling is known to interfere with the intrinsic apoptosis pathway, we combined various BH3 mimetic drugs with azacitidine in our previously established patient-derived xenograft model. We demonstrate that JMML cells require both MCL-1 and BCL-XL for survival, and that these proteins can be effectively targeted by azacitidine and BH3 mimetic combination treatment. In vivo azacitidine acts via downregulation of antiapoptotic MCL-1 and upregulation of proapoptotic BH3-only. The combination of azacitidine with BCL-XL inhibition was superior to BCL-2 inhibition in eliminating JMML cells. Our findings emphasize the need to develop clinically applicable MCL-1 or BCL-XL inhibitors in order to enable novel combination therapies in JMML refractory to standard therapy.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Venetoclax Combined with “7+3” Induction Chemotherapy Induces High Rates of MRD-Negative Remission in Newly Diagnosed AML Patients Fit for Intensive Chemotherapy Irrespective of Age

Introduction: Approximately 70% of patients (pts) with acute myeloid leukemia (AML) treated with the “7+3” regimen achieve complete remission (CR), with 30-50% of those attaining measurable residual disease (MRD) negative state by multiparameter flow cytometry (MFC). However, despite initial responses, the majority of pts eventually relapse. Venetoclax (Ven) added to intensive chemotherapy (IC) facilitates apoptosis of leukemic stem cells (LSC), and may result in deeper, more durable remissions. Moreover, IC induces MCL-1 downregulation and may overcome Ven resistance. We present safety and efficacy results from a phase 1b study of escalating cumulative Ven doses combined with daunorubicin (Dauno)+cytarabine (AraC) followed by high-dose cytarabine (HiDAC) chemotherapy in pts with newly diagnosed AML (NCT05342584). Objectives: The primary objective of this study is to determine the optimal dose of Ven in combination with Dauno & AraC and HiDAC chemotherapy. Secondary objectives are response per 2022 ELN, survival (OS), event-free survival (EFS) and duration of response (DoR). A key exploratory objective is to assess rates of LSC eradication by means of MFC and single cell DNA (Tapestri, Mission Bio) and RNA sequencing (“high-resolution MRD assay”). Methods: Eligible pts must have a new diagnosis of AML, be 18-75 yrs of age and deemed fit for intensive chemotherapy. In a 3+3 design, Ven in escalating duration (400 mg D1-8 or -11 or -14, including 2-day ramp-up) is combined with AraC 100mg/m2 (D2-8) and Dauno 60mg/m2 (D2-4) in 2 separate age groups (<60 and ≥60yrs); Dauno 90mg/m2 was initially studied in pts < 60yrs, but since 04/2023 the dose is no longer explored. During consolidation, fixed dose Ven (200 mg x7days) is used in combination with age-adjusted HiDAC (1.5g/m2 in <60yrs and 1g/m2 in ≥60yrs, every 12hrs on days 1,3,5). In the expansion phase, additional pts in each age group will undergo 1:1 randomization between the starting and highest tolerated dose determined during dose escalation, for a total of 20 pts per dose cohort in both phases. Results: Twenty-seven AML pts enrolled and 25 completed the induction phase to date, 2 are actively treated. Median age was 59yrs, 44% were females and 67% ethnic or racial minorities. Fourteen (52%) pts had poor-risk genotypes by ELN 2022. Fourteen pts were treated with Ven x 8d, 11 in Dauno60 and 3 in Dauno90 cohorts, 7 pts with Ven x11d and 6 pts with Ven x14d. No DLTs were observed, and the toxicity profile was similar to historical experience with the “7+3” regimen. There were no induction mortality deaths. Twenty pts (20/25, 80%) achieved a composite complete remission (CRc) with single induction (19 CRs and 1 CRi), 10/13 in <60 and 10/12 in ≥60yr groups. Median time to ANC≥0.5K/uL and PLT≥50K/uL was 26 days and time to CR was 32 days. Of those in CRc, all except three pts < 60yrs were MFC-MRD negative (17/20, 85%). Among 8 patients with available MRD PCR testing, 6 achieved molecular MRD-negative and 2 low-level molecular MRD CRs. With a median 5.6-month follow-up time, median EFS or OS have not been reached, 25/27 (93%) patients are alive (10/12 ≥60yrs), 12 pts have completed the protocol, 5 continue on therapy and 5 underwent allogeneic transplantation. From those in CRc, 7 younger and 8 older pts (75%) remain in remission without evidence of hematologic or molecular relapse. Of 5 pts with complex karyotype plus TP53 mutated AML, 3 did not respond and 2 had an early treatment failure. Table 1 summarizes patient characteristics, toxicity and outcomes. Conclusions: Ven 400mg in combination with “7+3” induction chemotherapy is safe and highly effective in achieving MRD negative remissions in the vast majority of patients. Responses were low in TP53 mutated pts, suggesting no benefit of the addition of venetoclax to intensive chemotherapy, similar to what has been observed when venetoclax is added to standard dose hypomethylating agents in those pts, for whom non-cytotoxic approaches are being explored. The study is completing enrollment in the Dauno60 + Ven x14d cohorts and is entering the expansion phase in order to better explore the optimal Ven dose to combine with the “7+3” regimen. Updated results, as well as rates of LSC elimination post-induction will be presented at the meeting.

Allosteric SHP2 inhibition increases apoptotic dependency on BCL2 and synergizes with venetoclax in FLT3- and KIT-mutant AML

Mutations in the receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although selective FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report the pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppresses proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that pharmacologic SHP2 inhibition unveils an Achilles' heel of RTK-driven AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in AML cell lines and in clinically relevant xenograft models. Our results provide mechanistic rationale and pre-clinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.

ATP1A1/BCL2L1 predicts the response of myelomonocytic and monocytic acute myeloid leukemia to cardiac glycosides

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

Discovery of Novel Fedratinib-Based HDAC/JAK/BRD4 Triple Inhibitors with Remarkable Antitumor Activity against Triple Negative Breast Cancer.

Multitarget HDAC inhibitors capable of simultaneously blocking the BRD4-LIFR-JAK1-STAT3 signaling pathway hold great potential for the treatment of TNBC and other solid tumors. Herein, novel Fedratinib-based multitarget HDAC inhibitors were rationally designed, synthesized, and biologically evaluated, among which compound 25ap stood out as a potent HDAC/JAK/BRD4 triple inhibitor. Satisfyingly, compound 25ap led to concurrent inhibition of HDACs and the BRD4-LIFR-JAK1-STAT3 signaling pathway, which was validated by hyper-acetylation of histone and α-tubulin, hypo-phosphorylation of STAT3, downregulation of LIFR, MCL-1, and c-Myc in MDA-MB-231 cells. The multitarget effects of 25ap contributed to its robust antitumor response, including potent antiproliferative activity, remarkable apoptosis-inducing activity, and inhibition of colony formation. Notably, 25ap possessed an acceptable therapeutic window between normal and cancerous cells, desirable in vitro metabolic stability in mouse microsome, and sufficient in vivo exposure via intraperitoneal administration. Additionally, the in vivo antitumor potency of 25ap was demonstrated in an MDA-MB-231 xenograft model.

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.