Steffan T. Nawrocki

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of RCC and is defined by loss of von Hippel–Lindau (VHL) function and dysregulated hypoxia signaling. Hyperactivation of the mTOR pathway is also frequently observed. These factors increase anabolic and protein-synthetic demand leading to proteotoxic stress. To survive under these conditions, ccRCC cells rely on adaptive endoplasmic reticulum (ER) stress responses and autophagy to maintain proteostasis. Regulated in Development and DNA Damage Response 1 (REDD1, also known as DDIT4) is a key stress response protein that integrates hypoxic, metabolic, and ER stress signals to regulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) activity and redox homeostasis. REDD1 can either suppress tumor growth through mTOR inhibition or promote survival by facilitating oxidative stress tolerance and autophagy depending on the specific context. In ccRCC, REDD1 expression is driven by VHL loss and HIF stabilization. Yet, mTORC1 activity frequently persists due to dysregulation of upstream repressors such as TSC1/TSC2, direct activating mutations in mTOR, and enhanced growth factor signaling. This paradox underscores the complex interplay between REDD1 and the oncogenic signaling landscape of RCC. Here, we review emerging evidence positioning REDD1 as a pivotal link between hypoxia, mTOR signaling, and ER stress in ccRCC and discuss how therapeutic modulation of this pathway through ER stress inducers, proteasome inhibitors, and p97/valosin-containing protein (VCP) inhibitors may overcome adaptive resistance and improve patient outcomes.

Epigenetic therapies can reverse malignant transcriptional programs, but their activity is often constrained by adaptive stress responses that allow cancer cells to survive and recover. Autophagy is a central component of this adaptation. By sustaining metabolic homeostasis, mitochondrial fitness, proteostasis, and lysosomal recycling, autophagy can buffer the cellular injury produced by inhibitors of DNA methyltransferases, histone deacetylases, and bromodomain proteins. These observations provide a strong rationale for combining epigenetic therapies with autophagy inhibition. However, the field has too often equated autophagosome accumulation with increased autophagic flux and treatment-induced autophagy with a functional dependency. Moreover, the lysosomotropic agents used in early clinical studies incompletely inhibit autophagy and exert broader effects on lysosomal biology. Consequently, compelling preclinical activity has not translated into consistent clinical benefit. Here, we critically evaluate the mechanisms through which epigenetic therapies engage autophagy, distinguish correlative markers from causal evidence of resistance, and assess the preclinical and clinical data supporting combination strategies. We also define the major barriers to translation, including inadequate pharmacodynamic assays, a lack of predictive biomarkers, context-dependent effects on tumor and immune cells, and the limited potency and selectivity of current autophagy inhibitors. Defining when, where, and how autophagy becomes a targetable dependency will be essential to translate this compelling biology into an effective precision treatment.

Acute myeloid leukemia (AML) remains defined by therapeutic resistance, adverse genetic heterogeneity, and poor long-term survival, underscoring the need for mechanistically informed regimens that exploit core stress-adaptation liabilities. Polo-like kinase 1 (PLK1) is a central regulator of replication recovery and mitotic progression, yet direct PLK1 inhibitors have been limited by tolerability and incomplete target suppression. Here, we define a clinically actionable strategy that functionally targets PLK1 by combining inhibition of the AAA+ ATPase p97/valosin-containing protein with the hypomethylating agent decitabine (DAC). Using AML cell lines, primary patient specimens, and an orthotopic in vivo model, we show that the clinically relevant p97 inhibitor CB-5339 induces proteotoxic and replication stress, activates the unfolded protein response, and triggers apoptosis. Formal synergy analyses demonstrate that CB-5339 and DAC cooperate across molecular subtypes, including FLT3-ITD+, KMT2A-rearranged, and TP53-mutant AML, to reduce cell viability at pharmacologically relevant concentrations. Transcriptomic, biochemical, and genetic studies identify synergistic suppression of PLK1 as a central consequence of combination treatment, converting stress-dependent PLK1 reliance into a therapeutic vulnerability. Consistent with this mechanism, PLK1 knockdown phenocopies the antileukemic effects of the combination and enhances sensitivity to both agents. In vivo, CB-5339/DAC is well tolerated, significantly prolongs survival, reduces leukemic burden, and suppresses PLK1 in bone marrow blasts. Together, these data establish p97 inhibition as a rational means to exploit replication and proteotoxic stress in AML and provide strong rationale for clinical evaluation of CB-5339 plus DAC in high-risk disease.Significance:This study defines a clinically actionable strategy to overcome therapeutic resistance in AML by functionally suppressing PLK1 through combined p97 inhibition and hypomethylating therapy. By exploiting convergent proteotoxic and replication stress, the regimen produces robust, well-tolerated antileukemic activity across high-risk genetic subtypes, supporting near-term clinical evaluation.

PLK1 is a key pharmacodynamic target of the CB-5339/DAC combination. A, Targeted PLK1 knockdown impairs AML cell proliferation. MOLM-13 cells were transduced with nontargeted control of PLK1-targeted lentiviral shRNA. Knockdown efficiency was determined by immunoblotting. Cell numbers were quantified for both conditions at 48 hours after lentiviral transduction. N = 3 ± SD; * indicates a significant difference from control shRNA, P < 0.005. B and C, Effects of targeted PLK1 knockdown on sensitivity to CB-5339 and DAC. MOLM-13 cells were infected with lentiviral nontargeted control or PLK1-targeted shRNA. Cells were treated with the indicated concentrations of CB-5339 and DAC. Cell viability was determined by MTT assay. N = 3 ± SD; * indicates a significant difference from control shRNA, P < 0.05. D, High PLK1 expression is associated with resistance to hypomethylating therapy. Within the ELN2022 adverse-risk group, patients with AML were stratified based on PLK1 levels comparing PLK1-low expression (bottom 25%) and PLK1-high expression (top 25%). Hypomethylating therapy response (azacitidine) is shown as an AUC in which higher values indicate greater resistance. Each dot represents an individual patient. Statistical significance was determined using a two-sided Wilcoxon rank-sum test (*, P = 0.05). E, PLK1 is a determinant of sensitivity to the CB-5339/DAC combination. MOLM-13 cells transduced with nontargeted control shRNA or PLK1-targeted shRNA lentiviral vectors were treated with 250 nmol/L CB-5339, 500 nmol/L DAC, or both drugs for 72 hours. The impact of drug treatment on cell viability was determined by MTT assay. N = 3 ± SD; * indicates a significant difference from control shRNA, P < 0.005. F, Overexpression of PLK1 promotes resistance to CB-5339 and DAC. PLK1 was overexpressed in MOLM-13 cells using PLK1-Myc-DDK lentiviral particles. Overexpression efficiency was assessed by immunoblotting. Vector control and PLK1-overexpressing cells were treated with 200 nmol/L CB-5339, 300 nmol/L DAC, and the combination for 72 hours. The effects of drug treatment on cell viability were quantified for each experimental condition by MTT assay. Mean ± SD, n = 3. * indicates a significant difference from vector control cells, P < 0.05.

Figure S3. CB-5339 synergizes with venetoclax (VEN) and DAC to reduce AML cell viability.

CB-5339 in combination with DAC prolongs survival and is well tolerated in an orthotopic AML xenograft model. A, The CB-5339/DAC combination significantly increases overall survival. A disseminated mouse model of AML was generated by injecting MV4-11 cells into the tail veins of NOG mice. Mice were treated with vehicle control, 50 mg/kg CB-5339 daily (oral), 2.5 mg/kg DAC twice weekly (intraperitoneally), or both drugs for the duration of the study. Overall survival for each group was determined by Kaplan–Meier analysis. N = 10 ± SD; * indicates a significant difference from control and single-agent groups, P < 0.005. Median survival values are as follows: vehicle = day 14, CB-5339 = day 19, DAC = day 18, and CB-5339 + DAC = day 22. B, The CB-5339/DAC combination is well tolerated. Mouse weight is shown for all treatment groups. N = 10 ± SD. C, Administration of the CB-5339/DAC combination diminishes CD33+ blasts and PLK1 levels in vivo. Bone marrow specimens were collected from mice in all experimental groups and subjected to IHC analysis to quantify PLK1 and CD33 (leukemia cell marker) levels. Scale bar, 50 μm. D, Quantification of IHC analyses. The number of CD33+ cells were counted in five random fields. For PLK1, signal intensity was quantified using ImageJ software. N = 5 ± SD; * indicates a significant difference from control and single-agent groups, P < 0.005. E, Schematic illustration of the pharmacodynamic synergy of the CB-5339/DAC combination.

Figure S1. 2D Synergy heatmaps of the CB-5339/DAC combination.

Transcriptomic analyses identify PLK1 as a top early downregulated pharmacodynamic (PD) target of the CB-5339/DAC combination. A, MOLM-13 and MV4-11 cells were treated with 500 nmol/L CB-5339, 1 μmol/L DAC, or both drugs for 6 hours (MV4-11) or 12 hours (MOLM-13) and subjected to RNA sequencing analyses. Time points were selected based on the kinetics of the PD response for each individual cell line to calibrate gene level effects. Heatmaps depict the top PD targets in each cell line. B, qRT-PCR validates the impact of treatment with the CB-5339/DAC combination on PLK1 expression. MV4-11 and MOLM-13 cells were treated with the combination of 250 nmol/L CB-5339, 500 nmol/L DAC for 0, 4, and 8 hours. Relative gene expression levels were quantified by TaqMan qRT-PCR and normalized to 18S rRNA. N = 3 ± SD; * indicates a significant difference from control, P < 0.005. C and D, The CB-5339/DAC combination effectively diminishes PLK1 protein levels. AML cell lines and primary AML specimens were treated with 250 nmol/L CB-5339, 500 nmol/L DAC, or both drugs for 24 hours. The impact of drug treatment on PLK1 protein levels was determined by immunoblotting. Actin documented equal protein loading. E,PLK1 expression is significantly upregulated in patients with AML with adverse-risk features. Patients with AML were stratified by ELN2022 risk group (favorable vs. adverse), and PLK1 expression was quantified. Each dot represents an individual patient. The center line indicates the median and boxes represent the interquartile range. Statistical significance was assessed using a two-sided Wilcoxon rank-sum test (*, P = 0.04).

Figure S2. CB-5339 and DAC synergize to trigger apoptosis.

Figure S4. Gene modulation overlap between MOLM-13 and MV4-11 cells.