ZBTB11 Depletion Targets Metabolic Vulnerabilities in K-Ras Inhibitor Resistant PDAC

Abstract

Over 95% of pancreatic ductal adenocarcinomas (PDAC) harbor oncogenic mutations in K-Ras. Upon treatment with K-Ras inhibitors, PDAC cancer cells undergo metabolic reprogramming towards an oxidative phosphorylation-dependent, drug-resistant state. However, direct inhibition of complex I is poorly tolerated in patients due to on-target induction of peripheral neuropathy. In this work, we develop molecular glue degraders against ZBTB11, a C₂H₂ zinc finger transcription factor that regulates the nuclear transcription of components of the mitoribosome and electron transport chain. Our ZBTB11 degraders leverage the differences in demand for biogenesis of mitochondrial components between human neurons and rapidly-dividing pancreatic cancer cells, to selectively target the K-Ras inhibitor resistant state in PDAC. Combination treatment of both K-Ras inhibitor-resistant cell lines and multidrug resistant patient-derived organoids resulted in superior anti-cancer activity compared to single agent treatment, while sparing hiPSC-derived neurons. Proteomic and stable isotope tracing studies revealed mitoribosome depletion and impairment of the TCA cycle as key events that mediate this response. Together, this work validates ZBTB11 as a vulnerability in K-Ras inhibitor-resistant PDAC and provides a suite of molecular glue degrader tool compounds to investigate its function.

Figure 1 |. OXPHOS is upregulated in K-Ras inhibitor resistant PDAC cells, and countered by ZBTB11 knockdown.

A. Sotorasib/AMG-510-resistant MIA PaCa-2 cells are 100-fold less sensitive to sotorasib/AMG-510 than parental MIA PaCa-2 cells. Cells were treated with the indicated dose of sotorasib for 72 hrs, and viability was evaluated by CellTiterGlo^®. Data is depicted as the average +/− standard deviation (S.D.) of n = 3 biological replicates and is normalized to DMSO vehicle treatment controls. See also Fig. S1A. B. Sotorasib-resistant MIA PaCa-2 cells perform higher basal and maximal levels of OXPHOS than parental MIA PaCa-2 cells. C. Sotorasib-resistant MIA PaCa-2 cells are more energetic than parental MIA PaCa-2 cells. D. Expression of genes regulated by ZBTB11 decreases following CRISPRi-mediated knockdown. Cells were treated with 500 ng/ml of doxycycline for 72 hrs to induce Cas9 expression, and mRNA levels were quantified by RT-qPCR. Data is depicted as the average +/− S.D. of n = 3 biological replicates and is normalized to H₂O vehicle treatment controls. See also Fig. S2. E. ZBTB11 knockdown reduces rates of OXPHOS. Cells were treated with 500 ng/ml of doxycycline for 72 hrs to induce Cas9 expression. F. ZBTB11 knockdown reduces ATP production by OXPHOS. B-C, E-F. Cellular respiration rates were evaluated in a mitochondrial stress test using a Seahorse analyzer. Oxygen Consumption Rates (OCR) and Extracellular Acidification Rates (ECAR) from the mitochondrial stress test were used to calculate ATP production rates. Data is depicted as the average +/− S.D. of n = 2 biological replicates with n = 3 technical replicates each.

Figure 2 |. Development of a ZBTB11 molecular glue degrader.

A. Depiction of ZBTB11 domains and the ZBTB11-HiBiT assay. B. Screening of glutarimide-containing analogues in the ZBTB11-HiBiT assay identifies candidate ZBTB11 degraders. MOLT-4 ZBTB11-HiBiT knock-in cells were treated with 10 μM compound for 8 hrs. Data is depicted as the average of n = 3 biological replicates and is normalized to DMSO vehicle treatment control. C. Chemical structure of screening hits and optimized ZBTB11 degrader and negative controls. D. JWJ-01–306, but not negative controls, degrades ZBTB11-HiBiT in MIA PaCa-2 knock-in cells. Cells were treated for 5 hrs with the indicated compound. E. Mechanism-based controls rescue ZBTB11-HiBiT degradation in MIA PaCa-2 knock-in cells. Cells were pre-treated with carfilzomib, MLN4924, or lenalidomide for 1 hr prior to treatment with the indicated compound for 5 hrs. Protein levels were quantified by western blot. Depicted blots are representative of n = 3 independent experiments. Uncropped blots found in Source Data. F. JWJ-01–306, but not negative controls, induces CRBN:degrader:ZBTB11 complex in NanoBRET^™ ternary complex assay. A MOLT-4 cell line with stable expression of the indicated constructs was generated. Cells were pre-treated with carfilzomib and the HaloTag^® NanoBRET^™ 618 Ligand for 1 hr prior to treatment with the indicated compound for 5 hrs. G. Computationally generated model of the CRBN:JWJ-01–306:ZBTB11 ZF10 complex reveals binding mode and key residues for complex stabilization. Top ten scoring structure clusters found in Source Data. H. Global proteomics analysis of MIA PaCa-2 cells treated with 10 μM JWJ-01–306 for 5 hrs. Samples were prepared as n = 3 biological replicates. Full datasets in Dataset 2. I. ZBTB11 K866T ablates ternary complex formation. MOLT-4 cell lines with stable expression of the indicated constructs were generated and used in the NanoBRET^™ ternary complex assay. J. ZBTB11 K866T rescues JWJ-01–306-mediated degradation. MIA PaCa-2 cell lines with stable expression of the indicated constructs were generated. Cells were treated with JWJ-01–306 for 5 hrs. K. K866 is necessary but not sufficient for ZBTB11 degradation. MOLT-4 cell lines with stable expression of the indicated constructs were generated. Cells were treated with 1 μM screening hit compound ALV-05–184 for 5 hrs. D, F, I-K. Data is depicted as the average +/− S.D. of n = 3 biological replicates and is normalized to DMSO vehicle treatment control.

Figure 3 |. JWJ-01–306 combination treatment overcomes acquired resistance to K-Ras inhibitors in PDAC via metabolic pathway reprogramming.

A. JWJ-01–306 synergizes with sotorasib to inhibit proliferation of sotorasib-resistant MIA PaCa-2 cells. Cells were treated with the indicated compounds and cell numbers were measured using trypan blue exclusion. Data is depicted as the average +/− S.D. of n = 3 biological replicates. B. JWJ-01–306 effectively inhibits proliferation of SUIT2 cells. Cells were treated with the indicated compounds and cell confluence was measured using a Cellcyte X live cell analyzer. Data is depicted as the average +/− S.D. of n = 3 biological replicates with n = 4 technical replicates each. C. Sotorasib-resistant MIA PaCa-2 cells were treated with DMSO, 1 μM sotorasib, 10 μM JWJ-01–306, or 1 μM sotorasib + 10 μM JWJ-01–306 (combo) for 72 hrs followed by global proteomics analysis, n = 3 biological replicates per condition. Protein abundance changes were analyzed by one-way ANOVA test (FDR < 0.05) and clustered using K-nearest neighbors clustering. Each line within the clusters is color-coded according to its distance from the cluster center, ranging from purple (close) to light blue (far). Proteins in each cluster were then evaluated for pathway enrichment using GO and KEGG pathway analysis. Representative pathways are shown and pathways associated with ZBTB11 function are highlighted in blue text. Full datasets in Dataset 4.

Figure 4 |. Metabolic reprogramming of K-Ras inhibitor resistant PDAC cells by ZBTB11 degradation.

A. ZBTB11 degradation reduces rates of OXPHOS in sotorasib-resistant MIA PaCa-2 cells. Cells were treated with 10 μM of the indicated compound for 24 hrs. B. ZBTB11 degradation reduces ATP production by OXPHOS in sotorasib-resistant MIA PaCa-2 cells. C. ZBTB11 degradation reduces rates of OXPHOS in MRTX1133-resistant SUIT2 cells. Cells were treated with 10 μM of the indicated compound for 24 hrs. D. ZBTB11 degradation reduces ATP production by OXPHOS in MRTX1133-resistant SUIT2 cells. E. Overexpression of ZBTB11^WT or ZBTB11 ^K866T rescues JWJ-01–306-mediated reduction of OXPHOS. Sotorasib-resistant MIA PaCa-2 cell lines with stable expression of NanoLuc-ZBTB11^WT or NanoLuc-ZBTB11^K866T were generated. Cells were treated with 10 μM JWJ-01–306 for 24 hrs. F. Overexpression of ZBTB11^WT or ZBTB11 ^K866T rescues JWJ-01–306-mediated reduction of ATP production by OXPHOS. G. ZBTB11 degradation induces downregulation of ZBTB11-regulated genes. Sotorasib-resistant MIA PaCa-2 cells were treated with 10 μM of the indicated compound for 24 hrs. H. Overexpression of ZBTB11^WT or ZBTB11 ^K866T rescues JWJ-01–306-mediated downregulation of ZBTB11-regulated genes. Cells were treated with 10 μM JWJ-01–306 for 24 hrs. I-M. JWJ-01–306 disrupts TCA cycle flux and induces reductive glutamine metabolism. Sotorasib-resistant MIA PaCa-2 cells were treated with DMSO or 10 μM JWJ-01–306 for 24 hrs. Cells were then labeled with ¹³C-labeled glucose or glutamine for an additional 24 hrs followed by global metabolomics analysis, n = 3 biological replicates per condition. A-F. Cellular respiration rates were evaluated in a mitochondrial stress test with the indicated drugs using a Seahorse analyzer. OCR and ECAR from the mitochondrial stress test were used to calculate ATP production rates. A-D. Data is depicted as the average +/− S.D. of n = 2 biological replicates with n = 3 technical replicates each. E-F. Data is depicted as the average +/− S.D. of n = 5 biological replicates with n = 3 technical replicates each. G-H. mRNA levels were quantified by RT-qPCR. Data is depicted as the average +/− S.D. of n = 3 biological replicates and is normalized to DMSO vehicle treatment controls. I-M. Metabolite abundance data is depicted as the average +/− S.D. of n = 6 biological replicates (3 replicates each from ¹³C-labeled glucose and glutamine). Mass isotopomer distribution data is depicted as the average +/− S.D. of n = 3 biological replicates. Significance level is marked with asterisks (two-tailed student’s t-test, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001).

Figure 5 |. ZBTB11 degradation spares neurons and deepens the response of PDAC patient-derived organoids to K-Ras inhibitors.

A. Mitochondrial activity and oxidative stress assays show differential pharmacology of Complex I and ZBTB11 perturbation. hiPSC-derived neurons were treated for 24 h with DMSO, 1 μM IACS 010759, 1 μM JWJ-01–306 or 1 μM JWJ-01–368. B. Neurotoxicity assays show differential pharmacology of Complex I and ZBTB11 perturbation. hiPSC-derived neurons were treated for 72 h with DMSO, 1 μM IACS 010759, 1 μM JWJ-01–306 or 1 μM JWJ-01–368. C. Proliferation assays show ZBTB11 + K-Ras inhibitor combination has superior antiproliferative effects in PDAC patient-derived organoids. Cells were treated with DMSO vehicle, 1 μM JWJ-01–306, 200 nM MRTX1133 or 1 μM JWJ-01–306 + 200 nM MRTX1133 (COMBO) and cellular confluence observed using brightfield imaging at the indicated timepoints. A.C. Data plotted as mean +/− S.D. of n = 3 biological replicates.