Pro-apoptotic Protein Noxa Research Articles

Gambogenic acid induces apoptosis via upregulation of Noxa in oral squamous cell carcinoma

Gambogenic acid (GNA), a bioactive compound derived from the resin of Garcinia hanburyi, has demonstrated significant antitumor properties. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unclear. This study aimed to elucidate the apoptotic effects of GNA on OSCC cell lines CAL-27 and SCC-15. Our results indicated that GNA induced apoptosis by upregulating the pro-apoptotic protein Noxa. Mechanistic investigations revealed that GNA treatment led to the generation of reactive oxygen species (ROS), which activated endoplasmic reticulum (ER) stress, culminating in cell apoptosis. Inhibition of ROS production and ER stress pathways significantly mitigated GNA-induced Noxa upregulation and subsequent apoptosis. Furthermore, in vivo studies using a murine xenograft model demonstrated that GNA administration effectively inhibited the growth of CAL-27 tumors. Collectively, these findings underscore GNA’s potential as a therapeutic agent for the treatment of OSCC.

Platinum (IV) drugs with cannabidiol inducing mitochondrial dysfunction and synergistically enhancing anti-tumor effects

Chemotherapy resistance is an insurmountable problem in clinical anticancer therapy. Although Oxaliplatin is an effective chemotherapeutic agent for the treatment of colorectal cancer (CRC), it still suffers from serious toxicities as well as drug resistance. In this work, three Oxaliplatin tetravalent platinum prodrugs(O1-O3) and three novel mixed ammine/amine analogs(C1-C3) were constructed, introducing cannabidiol with anti-tumor activity in their axial position. All Pt(IV) prodrugs exhibited potent antitumor effects in a variety of tumor cell lines, especially in HCT-116 cells, where complex O3 showed strong inhibitory effects with the half maximal inhibitory concentrations (IC50) value of 6.02 ± 0.69 μM and about 2.6 times higher than that of Oxaliplatin. Further studies revealed that complex O3 decreased cellular mitochondrial membrane potential in a concentration-dependent manner and enhanced reactive oxygen species (ROS) accumulation by decreasing the expression of catalase, superoxide dismutase 2 (SOD2) and superoxide dismutase 3 (SOD3). Complex O3 induces mitochondrial dysfunction and upregulates the pro-apoptotic protein Noxa, ultimately leading to severe DNA damage. The upregulation of Phosphorylated histone protein H2AX (γ-H2AX) expression is clear evidence. In addition, O3 inhibits the expression of RAD51 protein and prevents DNA damage repair, thus overcoming drug resistance. This strategy of combining bioactive molecules cannabidiol with platinum drugs to improve therapeutic efficacy and overcome drug resistance has been proven to be very effective and deserves further investigation.

Activation of GCN2 By HC-7366 Results in Significant Anti-Tumor Efficacy As Monotherapy and Overcomes Resistance Mechanisms When Combined with Venetoclax in AML

Venetoclax (anti-BCL2) is approved for elderly patients with acute myeloid leukemia (AML) in combination with hypomethylating agents such as azacitidine or decitabine. However, several resistance mechanisms arise, impeding responses in patients. These resistance mechanisms include TP53 or FLT3-ITD mutations, suppression of pro-apoptotic proteins NOXA or PUMA, increases in oxidative phosphorylation and energy metabolism, or elevated expression of S100A8/A9. Recent reports have highlighted that hyperactivation of the integrated stress response (ISR) pathway overcomes venetoclax resistance through NOXA activation and glycolytic activity inhibition. However, these studies used drugs that indirectly activate the ISR with other off-target effects. General Control Nonderepressible 2 (GCN2) is an ISR kinase that senses and responds to nutrient stress conditions, and activation of GCN2 results in antitumor activity. We are developing HC-7366, a first-in-class, first-in-human direct GCN2 activator, and are currently evaluating this agent in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in AML, and how it impacts venetoclax resistance. AML patients with TP53 mutations are known to have lower response rates to venetoclax.The in vivo efficacy of HC-7366 in both CDX and PDX models was independent of TP53 mutation, resulting in 100% complete response and 100% tumor growth inhibition in TP53-mutated MOLM-16 and KG-1 tumor models, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of the ISR as evidenced by increased expression of the ATF4 targets ASNS and PSAT1. A genome-wide CRISPR screen in KG-1 cells revealed that GCN2 sgRNAs were significantly enriched in HC-7366-treated cells, highlighting the importance of GCN2 activation in mediating the mechanism of action of HC-7366. Interestingly, BCL2 sgRNAs were significantly depleted in HC-7366 treated cells, suggesting a synthetic lethal relationship with BCL2 inhibitors, such as venetoclax, when combined with HC-7366. Indeed, in the MV4-11 FLT3-ITD mutant model (a differentiated subtype of AML that shows limited response to venetoclax), the combination of HC-7366 and venetoclax produced substantial benefit resulting in 26% tumor regression and enhanced activation of the ISR pathway. HC-7366 treatment also increased mRNA and protein levels of NOXA and PUMA while reducing mitochondrial respiration and glycolysis in a GCN2-dependent manner, leading to a low energetic state. Consistent with its impact on cellular energetics, global metabolomic analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with glycolysis and the TCA cycle. Additionally, HC-7366 reduced protein levels of S100A8/A9, also known to correlate with venetoclax resistance. Our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax. The potential of HC-7366 to counteract multiple known resistance mechanisms to venetoclax could provide a viable treatment option for patients with relapsed/refractory AML when used in combination with venetoclax.

NOXA Accentuates Apoptosis Induction by a Novel Histone Deacetylase Inhibitor.

Epigenetic modifiers of the histone deacetylase (HDAC) family are often dysregulated in cancer cells. Experiments with small molecule HDAC inhibitors (HDACi) have proven that HDACs are a vulnerability of transformed cells. We evaluated a novel hydroxamic acid-based HDACi (KH16; termed yanostat) in human pancreatic ductal adenocarcinoma (PDAC) cells, short- and long-term cultured colorectal cancer (CRC) cells, and retinal pigment epithelial cells. We show that KH16 induces cell cycle arrest and apoptosis, both time and dose dependently in PDAC and CRC cells. This is associated with altered expression of BCL2 family members controlling intrinsic apoptosis. Recent data illustrate that PDAC cells frequently have an altered expression of the pro-apoptotic BH3-only protein NOXA and that HDACi induce an accumulation of NOXA. Using PDAC cells with a deletion of NOXA by CRISPR-Cas9, we found that a lack of NOXA delayed apoptosis induction by KH16. These results suggest that KH16 is a new chemotype of hydroxamic acid HDACi with superior activity against solid tumor-derived cells. Thus, KH16 is a scaffold for future research on compounds with nanomolar activity against HDACs.

Abstract 3950: Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors

Abstract Background: Lung cancer is the leading cause of cancer deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for ~80% of all lung cancers. Up to 69% of NSCLC are mutated for the tumor suppressor gene TP53, and of these, 84% are missense mutations, where wild-type (WT) TP53 tumor suppressor function is lost and in most cases, emergent cancer-promoting oncogenic activities are acquired. Due to the poor draggability of mutated p53 protein, the focus of therapeutics targeting NSCLC tumors with oncogenic missense mutant p53 has been on vulnerabilities that are not present in normal cells expressing WT p53. One such vulnerability involves oncogenic mutant p53-dependent attenuation of the oxidative stress response. The ability of cancer cells to survive oxidative stress depends on the regulated synthesis and activity of cellular antioxidants, including glutathione (GSH). Proteasome inhibitors (PIs) are known to induce cellular stress at multiple levels, including proteotoxic, autophagic, and oxidative stress, but the clinical utility of PIs has been limited by lack of efficacy in patients with solid tumors, including unselected lung cancer patients. Methods and Results: We now show bortezomib (BTZ) and other PIs are preferentially cytotoxic in oncogenic mutant p53-expressing lung cancer cells in culture. Furthermore, BTZ-treated oncogenic mutant p53-expressing cells display elevated levels of reactive oxygen species and BTZ’s cytotoxic effects were rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ cytotoxicity. Upon BTZ treatment of oncogenic mutant p53-expressing NSCLC cells, but not in WT p53 expressing NSCLC cells, we also observed the transcriptional induction of pro-apoptotic BH3-only BCL-2 family protein NOXA, consistent with an apoptotic mechanism of BTZ-induced cell death. Furthermore, in combination with BTZ, the use of BH3-mimetics such as venetoclax (ABT-199) or navitoclax (ABT-263) to inhibit the function of pro-survival proteins BCL-2/BCL-XL synergistically enhanced BTZ cytotoxicity, but only in oncogenic mutant p53-expressing NSCLC cells. In vivo mouse xenograft studies demonstrated that combination treatments of BTZ + venetoclax or BTZ + carboplatin (a standard of care chemotherapy for NSCLC) significantly attenuated oncogenic mutant p53-expressing tumor growth. Conclusions: Our data suggest that the preferential cytotoxicity of BTZ, and potentially other PIs, in oncogenic mutant p53-expressing NSCLC cells depends on the induction of toxic oxidative stress leading to NOXA-dependent apoptosis. Thus, combining PIs with drugs that limit antioxidant responses and/or sensitize cells to NOXA-induced apoptosis (e.g., BH3-mimetics) should synergistically and selectively kill NSCLC cells expressing oncogenic mutant p53 and open novel therapeutic avenues for the treatment of NSCLC. Citation Format: Kranthi Kumar Chougoni, Victoria L. Neely, Boxiao Ding, Eziafa Oduah, Khanh Nguyen, Bin Hu, Jennifer Koblinski, Bradford Windle, Hisashi Harada, Steven Grossman. Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3950.

Knockout of Noxa with CRISPR/Cas9 Increases Host Resistance to Influenza Virus Infection

The influenza virus induces cellular apoptosis during viral propagation, and controlling this virus-induced apoptosis process has been shown to have significant antiviral effects. The proapoptotic BH3-only protein Noxa is a strong inducer of apoptosis that can be activated by this virus, suggesting that Noxa has the potential as an anti-influenza target. To assess the value of Noxa as an antiviral target, we utilized CRISPR/Cas9 technology to produce a Noxa-knockout cell line. We found that the knockout of Noxa resulted in a dramatic reduction in the cytopathic effect induced by the influenza virus. Moreover, Noxa knockout decreased the expression of influenza viral proteins (NP, M2, HA, and NS2). In addition, Noxa deficiency triggered a complete autophagic flux to weaken influenza virus-induced autophagosome accumulation, indicating that Noxa may be a promising antiviral target for controlling influenza virus infections.

Niclosamide Inhibits the Growth of Acute Lymphoblastic Leukemia Cells By Increasing Intracellular ROS and Activation of p53-PUMA/NOXA

Acute lymphoblastic leukemia (ALL) is a heterogeneous clonal malignancy originated from B- or T-cell lymphoid precursor cells. The prognosis is extremely poor in refractory or relapsed cases. Niclosamide is an FDA-approved oral anthelmintic drug with anti-cancer property. In the present study, we investigated the anti-leukemic effect of niclosamide in ALL cells in vitro and in vivo. ALL cell lines REH and NALM-6, and primary cells of patients with ALL were treated with niclosamide according to the concentration gradient, and then detected by trypan blue, MTS, and soft agar colony assays. The transcriptional and translational levels of apoptosis/anti-apoptosis-related genes were assessed by immunoblotting and quantitative real-time PCR. Flow cytometry was used to detect the effect of niclosamide on apoptosis of ALL cells, mitochondrial membrane potential, and intracellular specific reactive oxygen species (ROS) levels. For animal studies, we generated a mouse xenograft model with NOD prkdc-/-IL-2Rg-/- mice intravenously engrafted with NALM-6-luc cells, the NALM-6 cells infected with retrovirus expressing luciferase. Mice were treated with 25 mg/kg/day niclosamide or solvent twice a day intraperitoneally. The effect of niclosamide against leukemia cells in mice was dynamically evaluated by an in vivo imaging system. Flow cytometry was used to analyze the percentage of leukemia cells in spleens and bone marrows of sacrificed mice. The overall survival of mice was evaluated by Kaplan-Meier survival curve. In the cell viability assay, we found that niclosamide effectively inhibited REH, NALM-6 and primary ALL cells with IC50 values of 1.72, 3.58 and 1.36 μmol/L, respectively. Immunoblotting showed increased levels of pro-apoptotic proteins p53, PUMA, NOXA, cyto C and PARP, but decreased level of anti-apoptotic protein survivin in ALL cells treated with niclosamide in a dose- and time-dependent manner. Flow cytometry showed that niclosamide induced apoptosis and mitochondrial membrane potential in ALL cells. We also found that the level of ROS began to increase in cells treated by niclosamide for 2 hours, which reached the maximum at 8 hours. The treatment of ROS inhibitor N-acetylcysteine (NAC) attenuated niclosamide-induced apoptosis of REH and NALM-6 cells, which reduced by 18.12% and 8.02%, respectively. NAC also inhibited niclosamide-induced accumulation of intracellular p53/PUMA/NOXA proteins in these cells. In the animal study, in vivo imaging system showed significantly reduced tumor burdens in mice treated with niclosamide compared with controls, with bioluminescence signals of 15.53 and 4.04 on day 21, respectively. Mice treated with niclosamide survived longer than controls (32.5 days vs 26.5 days, p < 0.01), which had lower percentage of CD45+ CD19+ leukemia cells in their bone marrows and spleens, compared with the control group (61.10% vs 34.76% and 54.88% vs 26.40%, respectively). In the present study, we found that niclosamide potently inhibited the growth of ALL cells and induced apoptosis via increased levels of ROS and the activation of p53 signaling. Our findings suggest that niclosamide may be a promisingly potent agent for ALL therapy.

UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.

The hexameric AAA-ATPase valosin-containing protein (VCP) is essential for mitochondrial protein quality control. How VCP is recruited to mammalian mitochondria remains obscure. Here we report that UBXD8, an ER- and lipid droplet-localized VCP adaptor, also localizes to mitochondria and locally recruits VCP. UBXD8 associates with mitochondrial and ER ubiquitin E3 ligases and targets their substrates for degradation. Remarkably, both mitochondria- and ER-localized UBXD8 can degrade mitochondrial and ER substrates in cis and in trans. UBXD8 also associates with the TOM complex but is dispensable for translocation-associated degradation. UBXD8 knockout impairs the degradation of the pro-survival protein Mcl1 but surprisingly sensitizes cells to apoptosis and mitochondrial stresses. UBXD8 knockout also hyperactivates mitophagy. We identify pro-apoptotic BH3-only proteins Noxa, Bik, and Bnip3 as novel UBXD8 substrates and determine that UBXD8 inhibits apoptosis via degrading Noxa and restrains mitophagy via degrading Bnip3. Collectively, our characterizations reveal UBXD8 as the major mitochondrial adaptor of VCP and unveil its role in apoptosis and mitophagy regulation.

Pituitary adenomas evade apoptosis via noxa deregulation in Cushing's disease.

SUMMARYSporadic pituitary adenomas occur in over 10% of the population. Hormone-secreting adenomas, including those causing Cushing’s disease (CD), cause severe morbidity and early mortality. Mechanistic studies of CD are hindered by a lack of in vitro models and control normal human pituitary glands. Here, we surgically annotate adenomas and adjacent normal glands in 25 of 34 patients. Using single-cell RNA sequencing (RNA-seq) analysis of 27594 cells, we identify CD adenoma transcriptomic signatures compared with adjacent normal cells, with validation by bulk RNA-seq, DNA methylation, qRT-PCR, and immunohistochemistry. CD adenoma cells include a subpopulation of proliferating, terminally differentiated corticotrophs. In CD adenomas, we find recurrent promoter hypomethylation and transcriptional upregulation of PMAIP1 (encoding pro-apoptotic BH3-only bcl-2 protein noxa) but paradoxical noxa downregulation. Using primary CD adenoma cell cultures and a corticotroph-enriched mouse cell line, we find that selective proteasomal inhibition with bortezomib stabilizes noxa and induces apoptosis, indicating its utility as an anti-tumor agent.

Abstract 363: Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers

Abstract Background: The dichotomy of redox signaling in cancer cell fate decisions has emerged as a topic of importance over the years. In particular, our group has highlighted the role of superoxide (O2 .-)-mediated signaling in the acquisition of drug resistance and cancer survival. Mechanistically, this involves post-translational modification of proteins involved in oncogenic signaling, such as c-Myc and NF-κB, as well as apoptosis resistance like Bcl-2 at the serine 70 phosphorylation site. Here we present evidence that O2 .--mediated signaling stabilizes anti-apoptotic protein Mcl-1, a commonly amplified gene in human cancers and is implicated in resistance to venetoclax, the Bcl-2 specific BH3 mimetic. Methods: Intracellular O2 .- levels was increased either pharmacologically or by siRNA mediated knockdown of SOD1 in MOLM14, RPMI8226, OCI-AML3 hematopoietic cell lines. Cell viability and apoptosis were assessed by MTT and Annexin V/DAPI staining. Protein expression was determined by Western blot analysis. BH3 profiling of patient-derived CLL cells was used to assess mitochondrial priming. Results: Elevated O2 .- levels increased Mcl-1 stability through Thr163 phosphorylation of Mcl-1, which in turn reduced ubiquitination-dependent degradation. Furthemore, venetoclax resistant cells possessed higher O2 .- levels and increase in intracellular O2 .- levels promoted resistance to Mcl-1 inhibitor, S63845 through increased binding between Mcl-1 and pro-apoptotic proteins NOXA and Bak. Contrarily, scavenging of O2 .- re-sensitized venetoclax-resistant leukemia cells to venetoclax. Importantly, BH3 profiling revealed that increase in intracellular O2 .- levels decreased overall mitochondrial priming in primary CLL cells by promoting sequestration of pro-apoptotic proteins that specifically target Mcl-1. Conclusion: Oncogenic activity of O2 .- is mediated by increased Mcl-1 stability through Thr163 phosphorylation, which resulted in a reduction in overall mitochondrial priming and increased binding of Mcl-1 to NOXA and Bak. Scavenging O2 .- reduces Mcl-1 levels and sensitizes resistant cells to venetoclax-induced apoptosis thereby demonstrating that manipulation of the redox microenvironment could be a potential vulnerability to be exploited in venetoclax resistant cancers Citation Format: Jolin Xiao Lai, Stephen Jun Chong, Matthew Davids, Shazib Pervaiz. Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 363.

The BCL-2 inhibitor ABT-199/venetoclax synergizes with proteasome inhibition via transactivation of the MCL-1 antagonist NOXA

Enhanced expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) protein is frequent in cancer. Targeting of BCL-2 with the specific inhibitor ABT-199 (Venetoclax) has significant clinical activity in malignant diseases such as chronic lymphocytic leukemia and multiple myeloma. The small molecule drug ABT-199 mimics the pro-apoptotic BCL-2 homology domain 3 of BH3-only proteins and blocks the hydrophobic BC-groove in BCL-2. We have previously shown that ABT-199 synergizes with the proteasome inhibitor (PI) bortezomib in soft tissue sarcoma derived cells and cell lines to induce apoptosis. Synergistic apoptosis induction relies on the pore-forming effector BAX and expression of the pro-apoptotic BH3-only protein NOXA. Bortezomib augments expression of NOXA by blocking its proteasomal degradation. Interestingly, shown here for the first time, expression of NOXA is strongly enhanced by ABT-199 induced integrated stress response (ISR). ISR transcription factors ATF3 & ATF4 mediate transactivation of the BH3-only protein NOXA which specifically inhibits the anti-apoptotic MCL-1. Thus, NOXA potentiates the efficacy of the BCL-2 inhibitor ABT-199 by simultaneous inhibition of MCL-1. Hence, ABT-199 has a double impact by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivated NOXA. By preventing degradation of NOXA PIs synergize with ABT-199. Synergism of ABT-199 and PIs therefore occurs on several, previously unexpected levels. This finding should prompt clinical evaluation of combinatorial regimens in further malignancies.

MEK and MCL-1 sequential inhibition synergize to enhance rhabdomyosarcoma treatment

Targeted agents have emerged as promising molecules for cancer treatment, but most of them fail to achieve complete tumor regression or attain durable remissions due to tumor adaptations. We used dynamic BH3 profiling to identify targeted agents effectiveness and anti-apoptotic adaptations upon targeted treatment in rhabdomyosarcoma. We focused on studying the use of BH3 mimetics to specifically inhibit pro-survival BCL-2 family proteins, overwhelm resistance to therapy and prevent relapse. We observed that the MEK1/2 inhibitor trametinib rapidly depleted the pro-apoptotic protein NOXA, thus increasing MCL-1 availability. Indeed, we found that the MCL-1 inhibitor S63845 synergistically enhanced trametinib cytotoxicity in rhabdomyosarcoma cells in vitro and in vivo. In conclusion, our findings indicate that the combination of a BH3 mimetic targeting MCL-1 with trametinib improves efficiency on rhabdomyosarcoma by blocking tumor adaptation to treatment.

NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFβ/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth invivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.

BH3-Only Proteins Noxa and Puma Are Key Regulators of Induced Apoptosis.

Apoptosis is an evolutionarily conserved and tightly regulated cell death pathway. Physiological cell death is important for maintaining homeostasis and optimal biological conditions by continuous elimination of undesired or superfluous cells. The BH3-only pro-apoptotic members are strong inducers of apoptosis. The pro-apoptotic BH3-only protein Noxa activates multiple death pathways by inhibiting the anti-apoptotic Bcl-2 family protein, Mcl-1, and other protein members leading to Bax and Bak activation and MOMP. On the other hand, Puma is induced by p53-dependent and p53-independent apoptotic stimuli in several cancer cell lines. Moreover, this protein is involved in several physiological and pathological processes, such as immunity, cancer, and neurodegenerative diseases. Future heat shock research could disclose the effect of hyperthermia on both Noxa and BH3-only proteins. This suggests post-transcriptional mechanisms controlling the translation of both Puma and Noxa mRNA in heat-shocked cells. This study was also the chance to recapitulate the different reactional mechanisms investigated for caspases.

A Phase 1b/2 Study of Navtemadlin (KRT-232), a Murine Double Minute 2 Inhibitor, Combined with a BCR-ABL Tyrosine Kinase Inhibitor in Patients with Relapsed/Refractory, TP53WT, Ph+ Chronic Myeloid Leukemia

A Phase 1b/2 Study of Navtemadlin (KRT-232), a Murine Double Minute 2 Inhibitor, Combined with a BCR-ABL Tyrosine Kinase Inhibitor in Patients with Relapsed/Refractory, TP53WT, Ph+ Chronic Myeloid Leukemia

A Phase I Study of Pevonedistat, Azacitidine and Venetoclax for Patients with Relapsed/Refractory Acute Myelogenous Leukemia (AML)

Background:Outcomes of patients with relapsed/refractory AML (RR-AML) have remained poor. Therapy with venetoclax based combinations in this setting leads to CR/CRi rates of 21-49% (DiNardo CD et al. Am J Hematol 2018, Aldoss I et al. Haematologica 2018, Stahl M et al. Blood Adv 2020). Preclinical studies with BCL-2 inhibitors indicate potential mechanisms of drug resistance including overexpression of the anti-apoptotic protein MCL-1 (Konopleva et al. Cancer Cell. 2006). Pevonedistat is a first in class inhibitor of Nedd8 activating enzyme that induces the pro-apoptotic protein NOXA leading to neutralization of MCL-1 and apoptosis. Preclinical studies evaluating the combination of pevonedistat and venetoclax against AML cell lines have demonstrated a synergistic effect (Knorr KL et al. Cell Death Differ. 2015). Hence, we designed a phase I study to assess the safety and tolerability of adding pevonedistat to the combination of azacitidine and venetoclax in patients with RR-AML.Study design and methods:We conducted a phase I study with the combination of pevonedistat, venetoclax and azacitidine in patients with RR-AML. Patients aged 18 years or above with morphologically documented RR-AML, ECOG performance status 0-2 and adequate organ function were eligible. Major exclusion criteria were isolated extramedullary relapse, hematopoietic cell transplantation (HCT) within 100 days of enrollment, and active acute GVHD. Previous therapy with hypomethylating agents (HMA) or venetoclax was not an exclusion criterion. The dose escalation phase was conducted using 3+3 design. Treatment included azacitidine (75 mg/m 2 daily x 7 days), venetoclax (400 mg daily x 28 days), and pevonedistat in escalating doses (10-20 mg/m 2 IV days 1,3,5 of each cycle) with a cycle length of 28 days. Pevonedistat was given at 10 mg/m 2 dose in cohort 1, 15 mg/m 2 dose in cohort 2 and 20 mg/m 2 dose in cohort 3.The primary endpoint is to determine the recommended phase 2 dose (RP2D) and toxicity profile of pevonedistat, azacitidine and venetoclax. Other endpoints included determination of response rates, duration of response, survival, pharmacokinetics, correlation of response rates with AML genomic profile, correlation of pretreatment levels of BCL2, BCLXL, MCL1, BAX or BAK with response, determination of changes in NOXA (PMAIP1) mRNA and protein expression pre-and post-pevonedistat treatment, evaluation of BH3 mimetic profiling on bone marrow samples by flow cytometry and assessing the sensitivity of leukemia and leukemic stem/progenitor cells to pevonedistat ex vivo.Results:Thirteen patients participated in the dose escalation phase, 12 of whom were evaluable. Median age was 69 years (61-91), 30.8% had secondary/therapy related AML, 69.2% with adverse risk disease, 53.8% previously received venetoclax/HMA and 23.1% had relapse after prior allogeneic hematopoietic cell transplantation (HCT) (Table 1). Seven patients were enrolled into cohort 1 (pevonedistat 10 mg/m 2 dose) of which one was not evaluable, three patients enrolled into cohort 2 (pevonedistat 15 mg/m 2 dose), and three patients enrolled into cohort 3 (pevonedistat 20 mg/m 2 dose). Grade 3 or higher AEs included febrile neutropenia (23%), infection (15%), anemia (38%), neutropenia (54%), thrombocytopenia (38%). There was 1 dose limiting toxicity (DLT) in cohort 1 (atrial fibrillation) that triggered cohort expansion. However, subsequent patients did not experience DLT despite planned dose escalation. Of the 12 evaluable total patients, CR/CRi was observed in 5 (41.6%) patients. Notably, patients with RR-AML who were venetoclax/HMA naïve had a CR/CRi 83.3% (5/6 patients). The response rates for each cohort are summarized in Table 2. Three of the five patients with CR/CRi (60%) achieved MRD negativity by flow cytometry. Four patients who achieved CR/CRi proceeded to allogeneic HCT after therapy. Median OS of the cohort was 5.4 months (1.8-14) and median OS was not reached in patients with CR/CRi.Conclusions:The addition of pevonedistat to venetoclax and azacitidine backbone is safe and well tolerated in patients with RR-AML. Dose escalation yielded encouraging efficacy in venetoclax/HMA naïve RR-AML patients. The study is currently in the dose expansion phase. Details on correlative studies examining mechanisms of therapeutic efficacy and resistance will be reported in the main meeting. [Display omitted] DisclosuresGuru Murthy: Cardinal health: Honoraria; TG Therapeutics: Other: Advisory board meeting; DAVA Oncology: Honoraria; CancerExpertNow: Honoraria; Qessential: Honoraria; Techspert: Consultancy; Curio Sciences: Honoraria; Guidepoint: Consultancy. Abedin: AltruBio: Research Funding; Helsinn: Research Funding; Amgen: Honoraria; Pfizer: Research Funding; Agios: Honoraria; Actinium: Research Funding; Astellas Pharma Inc.: Research Funding. Litzow: Jazz: Other: Advisory Board; Pluristem: Research Funding; Actinium: Research Funding; Amgen: Research Funding; Astellas: Research Funding; AbbVie: Research Funding; Omeros: Other: Advisory Board; Biosight: Other: Data monitoring committee. Atallah: Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Honoraria, Speakers Bureau; Takeda: Consultancy, Research Funding; Abbvie: Consultancy, Speakers Bureau; Amgen: Consultancy.

Three novel piperidones exhibit tumor-selective cytotoxicity on leukemia cells via protein degradation and stress-mediated mechanisms

BackgroundCancer is an ongoing worldwide health problem. Although chemotherapy remains the mainstay therapy for cancer, it is not always effective and has detrimental side effects. Here, we present piperidone compounds P3, P4, and P5 that selectively target cancer cells via protein- and stress-mediated mechanisms.MethodsWe assessed typical apoptotic markers including phosphatidylserine externalization, caspase-3 activation, and DNA fragmentation through flow cytometry. Then, specific markers of the intrinsic pathway of apoptosis including the depolarization of the mitochondria and the generation of reactive oxygen species (ROS) were investigated. Finally, we utilized western blot techniques, RT-qPCR, and observed the cell cycle profile after compound treatment to evaluate the possible behavior of these compounds as proteasome inhibitors. For statistical analyses, we employed the one-way ANOVA followed by Bonferroni post hoc test.ResultsP3, P4, and P5 induce cytotoxic effects towards tumorigenic cells, as opposed to non-cancerous cells, at the low micromolar range. Compound treatment leads to the activation of the intrinsic pathway of apoptosis. The accumulation of poly-ubiquitinated proteins and the pro-apoptotic protein Noxa, both typically observed after proteasome inhibition, occurs after P3, P4, and P5 treatment. The stress-related genes PMAIP1, ATF3, CHAC1, MYC, and HMOX-1 were differentially regulated to contribute to the cytotoxic activity of P3–P5. Finally, compound P5 causes cell cycle arrest at the G2/M phase.ConclusionTaken together, compounds P3, P4, and P5 exhibit strong potential as anticancer drug candidates as shown by strong cytotoxic potential, activation of the intrinsic pathway of apoptosis, and show typical proteasome inhibitor characteristics.

Interactions of multidomain pro-apoptotic and anti-apoptotic proteins in cancer cell death.

Cancer is a global public health concern that is characterized by the uncontrolled growth of tumor cells. It is regarded as the subsequent cause of death after cardiovascular disease. The most common types of cancer include breast, colorectal, lung, and prostate. The risk factors attributed to the development of common types of cancer are tobacco smoking, excessive alcohol consumption, dietary factors, ultraviolet radiation (UV), and lack of physical activities. Two major cellular apoptotic pathways targeted in cancer therapies are intrinsic and extrinsic. These two pathways are regulated by different types of proteins, the multidomain pro-apoptotic proteins (Bak, Bax, and Bok), BH3-only pro-apoptotic proteins (Bid, Bim, Bad, Noxa, and Puma), and the anti-apoptotic proteins (Mcl-1, Bfl-1, Bcl-XL, Bcl-2, Bcl-w, and Bcl-B). Other significant molecules/factors that are known to execute cellular apoptotic pathways include bioactive compounds, and reactive oxygen species (ROS). Proteolytic caspases are known to play a vital role in the initiation of apoptotic activities in cancerous cells. Based on their functions, they are categorized into initiators and executioners. Nanotechnology has produced novel outcomes in modern medicine. The green synthesis of nanoparticles has demonstrated prospective improvements in cancer therapies in combination with the existing therapies including photodynamic therapy. This review aims at highlighting the association between pro-apoptotic and anti-apoptotic proteins, and their significance in cancer therapy.

Autophagic degradation of NOXA underlies stromal cell-mediated resistance to proteasome inhibitors in mantle cell lymphoma

Mantle cell lymphoma (MCL) is usually resistant to the current standard-of-care regimens and also to novel agents such as the proteasome inhibitor bortezomib. A better prognosis of leukemic variants of MCL suggests that MCL cells acquire drug resistance in nodal and/or bone marrow microenvironments via interaction with supporting cells. Bortezomib exerts cytotoxic action in MCL cells via stabilization of the pro-apoptotic BCL-2 family protein NOXA. Here we show that autophagic degradation of NOXA is a mechanism of bortezomib resistance in MCL cells in a tumor microenvironment. First, we demonstrated that interaction with bone marrow-derived or nodal stromal cells conferred bortezomib resistance to MCL cells in vitro and in a murine model. Co-culture of MCL cells with stromal cells enhanced bortezomib-induced ubiquitination and subsequent binding of NOXA to the p62 adaptor, which escorted NOXA to the lysosome for autophagic degradation. Finally, we found that not only direct contact with stromal cells but also stroma-derived humoral factors, especially interleukin-6, promoted selective autophagy and NOXA degradation in MCL cells. Targeting protective autophagy, for example, using the lysosome inhibitor chloroquine, might increase the efficacy of bortezomib-containing regimens in MCL.

Abstract 976: Inhibition of BCL-2 (by APG-2575) and CDK4/6 synergistically induces cell cycle arrest and apoptosis in ER+ breast cancer

Abstract Estrogen receptor positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) tumors represent the most common subset (about 75%) of all breast cancer cases. Combination treatment with endocrine therapy and a CDK4/6 inhibitor (CDK4/6i) is now the standard of care in early metastatic ER+ breast cancer. However, relapse or resistance to such combination therapy nearly inevitably occurs. Antiapoptotic protein BCL-2 is overexpressed in most cases of primary and metastatic ER+ breast cancer. We have shown that clinical-stage BCL-2 selective inhibitor APG-2575 enhances antitumor activity when combined with palbociclib in ER+ breast cancer xenograft models, including malignancies that are resistant to tamoxifen or progress after CDK4/6i treatment. We investigated mechanisms of action (MOAs) for synergistic effects of this combination. In MCF-7 cells sensitive to treatment with tamoxifen/fulvestrant, the CDK4/6i palbociclib inhibited expression of both ER and phospho-Rb (pRb) proteins, resulting in cell cycle arrest and senescence as demonstrated by cell cycle analysis and beta-galactosidase staining, respectively. Palbociclib did not initiate apoptosis as measured by TUNEL assays. In contrast, APG-2575 not only disrupted BCL-2:BIM complexes in MCF-7 cells but also blocked cell cycle (G1 to S) progression by suppressing expression of pRB, ER, and cyclin D1 proteins. APG-2575 also increased expression of proapoptotic proteins BIM and Noxa, as well as antiapoptotic protein MCL-1. APG-2575 also led to an increase in complexes of BIM with MCL-1, which is known to confer resistance against BCL-2 inhibitors. Interestingly, the combination treatment weakened induction of MCL-1 protein and reduced MCL-1:BIM complexes. Thus, the combination treatment synergistically triggered breast cancer cell apoptosis, as evidenced by increased TUNEL signal and cleavage of poly (ADP-ribose) polymerase 1 protein (a hallmark of apoptosis). Taken together, palbociclib drives breast cancer cells into a senescent state, and APG-2575 potentiates this antitumor effect by inducing cellular apoptosis. When combined with APG-2575, palbociclib inhibits MCL-1 expression and MCL-1:BIM complex formation as results of BCL-2 inhibition. Therefore, palbociclib reduces the threshold for apoptosis, while APG-2575 induces apoptosis by disrupting BCL-2:BIM complexes as well as enhancing expression of BIM and Noxa. Similar to palbociclib, APG-2575 also causes cell cycle arrest, and both agents collaboratively induce apoptosis in the combination setting. Our data reveal a viable MOA for synergistic effects, strengthening the scientific rationale for clinical development of the combination of the BCL-2 selective inhibitor APG-2575 and CDK4/6i palbociclib-based therapy in patients with ER+/HER2− breast cancer. Citation Format: Jing Deng, Ran Tao, Douglas D. Fang, Qiuqiong Tang, Yan Yin, Kaixiang Zhang, Yuanbao Li, Guoqin Zhai, Qixin Wang, Dajun Yang, Yifan Zhai. Inhibition of BCL-2 (by APG-2575) and CDK4/6 synergistically induces cell cycle arrest and apoptosis in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 976.