HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer.

BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome ß5 + ß2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits ß5 + ß1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.

High-dose carfilzomib achieves superior anti-tumor activity over low-dose and recaptures response in relapsed/refractory multiple myeloma resistant to lowdose carfilzomib by co-inhibiting the ß2 and ß1 subunits of the proteasome complex.

Optimal carfilzomib dosing is a matter of debate. We analyzed the inhibition profiles of proteolytic proteasome subunits ß5, ß2 and ß1 after low-dose (20/27 mg/m2) versus high-dose (≥36 mg/m2) carfilzomib in 103 pairs of peripheral blood mononuclear cells from patients with relapsed/refractory (RR) multiple myeloma (MM). ß5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas ß2 and ß1 were co-inhibited only by 36 and 56 mg/m2, respectively. Coinhibition of ß2 (P=0.0001) and ß1 activity (P=0.0005) differed significantly between high-dose and low-dose carfilzomib. Subsequently, high-dose carfilzomib showed significantly more effective proteasome inhibition than low-dose carfilzomib in vivo (P=0.0003). We investigated the clinical data of 114 patients treated with carfilzomib combinations. High-dose carfilzomib demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose carfilzomib. Therefore, we escalated the carfilzomib dose to ≥36 mg/m2 in 16 patients who progressed during low-dose carfilzomib-containing therapies. High-dose carfilzomib recaptured response (≥ partial remission) in nine (56%) patients with a median PFS of 4.4 months. Altogether, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose carfilzomib differs from that of low-dose carfilzomib by coinhibition of ß2 and ß1 proteasome subunits and, consequently, high-dose carfilzomib achieves a superior anti-MM effect than low-dose carfilzomib and recaptures the response in RRMM resistant to low-dose carfilzomib. The optimal carfilzomib dose should be ≥36 mg/m2 to reach a sufficient anti-tumor activity, while the balance between efficacy and tolerability should be considered in each patient.

Improving the efficacy of proteasome inhibitors in the treatment of renal cell carcinoma by combination with the human immunodeficiency virus (HIV)-protease inhibitors lopinavir or nelfinavir.

OBJECTIVES: To assess the potential of second-generation proteasome inhibition by carfilzomib and its combination with the human immunodeficiency virus (HIV) protease inhibitors (HIV-PIs) lopinavir and nelfinavir in vitro for improved treatment of clear cell renal cell cancer (ccRCC). MATERIALS AND METHODS: Cytotoxicity, reactive oxygen species (ROS) production, and unfolded protein response (UPR) activation of proteasome inhibitors, HIV-PIs, and their combination were assessed in three cell lines and primary cells derived from three ccRCC tumours by MTS assay, flow cytometry, quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. Proteasome activity was determined by activity based probes. Flow cytometry was used to assess apoptosis by annexin V/propidium iodide assay and ATP-binding cassette sub-family B member 1 (ABCB1) activity by MitoTracker™ Green FM efflux assay (Thermo Fisher Scientific, MA, USA). RESULTS: Lopinavir and nelfinavir significantly increased the cytotoxic effect of carfilzomib in all cell lines and primary cells. ABCB1 efflux pump inhibition, induction of ROS production, and UPR pre-activation by lopinavir were identified as underlying mechanisms of this strong synergistic effect. Combined treatment led to unresolved protein stress, increased activation of pro-apoptotic UPR pathway, and a significant increase in apoptosis. CONCLUSION: The combination of the proteasome inhibitor carfilzomib and the HIV-PIs lopinavir and nelfinavir has a strong synergistic cytotoxic activity against ccRCCin vitro at therapeutically relevant drug concentrations. This effect is most likely explained by synergistic UPR triggering and ABCB1-modulation caused by HIV-PIs. Our findings suggest that combined treatment of second-generation proteasome inhibitors and HIV-PIs should be investigated in patients with metastatic RCC within a clinical trial.

MiR-338-5p sensitizes glioblastoma cells to radiation through regulation of genes involved in DNA damage response.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with an extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to the treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain-specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and miR-338-5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased cell proliferation, increased cell cycle arrest, and apoptosis in comparison to irradiation-only cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, and ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation, and cell cycle regulation. To our knowledge, this is the first study to describe the role of miR-338-5p in GBM and its potential to improve the sensitivity of GBM to radiation.

Risk Score based on microRNA expression signature is independent prognostic classifier of glioblastoma patients.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. The prognosis of GBM patients varies considerably and the histopathological examination is not sufficient for individual risk estimation. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and were repeatedly proved to play important roles in pathogenesis of GBM. In our study, we performed global miRNA expression profiling of 58 glioblastoma tissue samples obtained during surgical resections and 10 non-tumor brain tissues. The subsequent analysis revealed 28 significantly deregulated miRNAs in GBM tissue, which were able to precisely classify all examined samples. Correlation with clinical data led to identification of six-miRNA signature significantly associated with progression free survival [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.33-2.94, P < 0.001] and overa+ll survival (HR 2.86, 95% CI 1.91-4.29, P < 0.001). O(6)-methylguanine-DNA methyltransferase methylation status was evaluated as reference method and Risk Score based on six-miRNA signature indicated significant superiority in prediction of clinical outcome in GBM patients. Multivariate Cox analysis indicated that the Risk Score based on six-miRNA signature is an independent prognostic classifier of GBM patients. We suggest that the Risk Score presents promising prognostic algorithm with potential for individualized treatment decisions in clinical management of GBM patients.

MicroRNAs involved in chemo- and radioresistance of high-grade gliomas.

High-grade gliomas (HGGs) are malignant primary brain tumors of glial cell origin. Despite optimal course of treatment, including maximal surgical resection followed by adjuvant chemo- and/or radiotherapy, the prognosis still remains poor. The main reason is the commonly occurring chemo- and radioresistance of these tumors. In recent years, several signaling pathways, especially PI3K/AKT and ATM/CHK2/p53, have been linked to the resistance of gliomas. Moreover, additional studies have shown that these pathways are significantly regulated by microRNAs (miRNAs), short endogenous RNA molecules that modulate gene expression and control many biological processes including apoptosis, proliferation, cell cycle, invasivity, and angiogenesis. MiRNAs are not only highly deregulated in gliomas, their expression signatures have also been shown to predict prognosis and therapy response. Therefore, they present promising biomarkers and therapeutic targets that might overcome the resistance to treatment and improve prognosis of glioma patients. In this review, we summarize the current knowledge of the functional role of miRNAs in gliomas resistance to chemo- and radiotherapy.

MiR-210 expression in tumor tissue and in vitro effects of its silencing in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3 % of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P < 0.003)). Increased expression levels of miR-210 in RCC tumor tissue were independently validated. We observed decreased viability of ACHN and CAKI-2 cells and accumulation of CAKI-2 in G2 phase of cell cycle after silencing of miR-210 expression. Downregulation of miR-210 also reduced the migratory and invasive potential of ACHN metastatic RCC cells. Moreover, we showed downregulation of HIF1a protein in both cell lines after miR-210 silencing indicating participation of miR-210 in hypoxic processes of RCC not only through regulation of its target mRNAs but also by indirect regulation of HIF1a. To our knowledge, this is the first report to show miR-210 regulatory effects on cell migration, invasive potential, and HIF1a protein in RCC cells.

The cytotoxic activity of carfilzomib together with nelfinavir is superior to the bortezomib/nelfinavir combination in non-small cell lung carcinoma.

Chemotherapy resistance is still a major problem in the treatment of patients with non-small-cell-lung carcinoma (NSCLC), and novel concepts for the induction of cytotoxicity in NSCLC are highly warranted. Proteotoxicity, the induction of cytotoxicity by targeting the ubiquitin proteasome system, represents an appealing innovative strategy. The combination of the proteasome inhibitor bortezomib (BTZ) and the proteotoxic stress-inducing HIV drug nelfinavir (NFV) synergistically induces proteotoxicity and shows encouraging preclinical efficacy in NSCLC. The second-generation proteasome inhibitor carfilzomib (CFZ) is superior to BTZ and overcomes BTZ resistance in multiple myeloma patients. Here, we show that CFZ together with NFV is superior to the BTZ + NFV combination in inducing endoplasmic reticulum stress and proteotoxicity through the accumulation of excess proteasomal substrate protein in NSCLC in vitro and ex vivo. Interestingly, NFV increases the intracellular availability of CFZ through inhibition of CFZ export from NSCLC cells that express multidrug resistance (MDR) protein. Combining CFZ with NFV may therefore represent a future treatment option for NSCLC, which warrants further investigation.

Identification and functional screening of microRNAs highly deregulated in colorectal cancer.

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

Clinical correlations of miR-21 expression in colorectal cancer patients and effects of its inhibition on DLD1 colon cancer cells.

PURPOSE: MicroRNA-21 (miR-21) is one of the miRNAs that are frequently and highly overexpressed in tumor tissue of colorectal cancer (CRC) patients; however, only a little is known about its functional role in CRC. METHODS: We examined the expression level of miR-21 in 44 paired samples of tumoral and non-tumoral colon tissues diagnosed for CRC using TaqMan real-time PCR method. Furthermore, we used miR-21 inhibitor (anti-miR-21) to transient knockdown of miR-21 in DLD-1 colon cancer cells and examined the effects of miR-21 silencing on viability, apoptosis, chemosensitivity, cell cycle, and migration of DLD1 cells. RESULTS: The expression levels of miR-21 were significantly increased in CRC tumor tissue (P < 0.0001). Significant differences in miR-21 levels were observed also between CRC tissues of patients with CRC in different clinical stages: I vs. II (P = 0.033) and I vs. IV (P = 0.021). Kaplan-Meier analysis proved that the miR-21 expression levels are correlated to shorter overall survival of CRC patients (P = 0.0341). MiR-21 silencing in DLD1 cell line had no effect on the cell viability; however, when combined with chemotherapeutics (5-FU, L-OHP, and SN38), it contributed to the decrease of cell viability. Suppression of miR-21 decreased cell migration ability of DLD-1 cells by nearly 30 % (P = 0.016). CONCLUSION: We have confirmed the overexpression of miR-21 in CRC samples and its correlation with advanced disease and shorter overall survival. These findings could be described in part by the fact that CRC cells with increased expression of miR-21 have higher migration ability.

Contribution of the Tumor Microenvironment to Metabolic Changes Triggering Resistance of Multiple Myeloma to Proteasome Inhibitors.

Virtually all patients with multiple myeloma become unresponsive to treatment with proteasome inhibitors over time. Relapsed/refractory multiple myeloma is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations, diverse proteomic and metabolic alterations, and profound changes of the bone marrow microenvironment. However, the molecular mechanisms that drive resistance to proteasome inhibitors within the context of the bone marrow microenvironment remain elusive. In this review article, we summarize the latest knowledge about the complex interaction of malignant plasma cells with its surrounding microenvironment. We discuss the pivotal role of metabolic reprograming of malignant plasma cells within the tumor microenvironment with a subsequent focus on metabolic rewiring in plasma cells upon treatment with proteasome inhibitors, driving multiple ways of adaptation to the treatment. At the same time, mutual interaction of plasma cells with the surrounding tumor microenvironment drives multiple metabolic alterations in the bone marrow. This provides a tumor-promoting environment, but at the same time may offer novel therapeutic options for the treatment of relapsed/refractory myeloma patients.

Mutant TP53 driving the Warburg Effect in Mantle Cell lymphoma.

The p53 mutation R273H in tumor cells leads to increased glucose uptake, lactic acidosis, and accelerated tumor growth, as was previously shown in mice. We here present a patient with mantle cell lymphoma harboring this p53_R273H mutation, whose clinical course is characterized by severe lactic acidosis, hypoglycemia, and aggressive disease.

Immunoproteasome Activity in Chronic Lymphocytic Leukemia as a Target of the Immunoproteasome-Selective Inhibitors.

Targeting proteasome with proteasome inhibitors (PIs) is an approved treatment strategy in multiple myeloma that has also been explored pre-clinically and clinically in other hematological malignancies. The approved PIs target both the constitutive and the immunoproteasome, the latter being present predominantly in cells of lymphoid origin. Therapeutic targeting of the immunoproteasome in cells with sole immunoproteasome activity may be selectively cytotoxic in malignant cells, while sparing the non-lymphoid tissues from the on-target PIs toxicity. Using activity-based probes to assess the proteasome activity profile and correlating it with the cytotoxicity assays, we identified B-cell chronic lymphocytic leukemia (B-CLL) to express predominantly immunoproteasome activity, which is associated with high sensitivity to approved proteasome inhibitors and, more importantly, to the immunoproteasome selective inhibitors LU005i and LU035i, targeting all immunoproteasome active subunits or only the immunoproteasome ß5i, respectively. At the same time, LU102, a proteasome ß2 inhibitor, sensitized B-CLL or immunoproteasome inhibitor-inherently resistant primary cells of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, multiple myeloma and plasma cell leukemia to low doses of LU035i. The immunoproteasome thus represents a novel therapeutic target, which warrants further testing with clinical stage immunoproteasome inhibitors in monotherapy or in combinations.

Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma.

PURPOSE: Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms. EXPERIMENTAL DESIGN: Response to vismodegib was evaluated in a cohort of 148 laBCC patients. Comprehensive genomic and transcriptomic profiling was performed in a subset of five intrinsically resistant BCC (IR-BCC). RESULTS: We identified that IR-BCC represents 6.1% of laBCC in the studied cohort. Prior treatment with chemotherapy was associated with IR. Genetic events that were previously associated with acquired resistance (AR) in BCC or medulloblastoma were observed in three out of five IR-BCC. However, IR-BCCs were distinct by highly rearranged polyploid genomes. Functional analyses identified hyperactivation of the HIPPO-YAP and WNT pathways at RNA and protein levels in IR-BCC. In vitro assay on the BCC cell line further confirmed that YAP1 overexpression increases the cell proliferation rate. CONCLUSIONS: IR to vismodegib is a rare event in laBCC. IR-BCCs frequently harbor resistance mutations in the Hh pathway, but also are characterized by hyperactivation of the HIPPO-YAP and WNT pathways.

Novel Peptide-drug Conjugate Melflufen Efficiently Eradicates Bortezomib-resistant Multiple Myeloma Cells Including Tumor-initiating Myeloma Progenitor Cells.

Introduction of the proteasome inhibitor bortezomib has dramatically improved clinical outcomes in multiple myeloma. However, most patients become refractory to bortezomib-based therapies. On the molecular level, development of resistance to bortezomib in myeloma cells is accompanied by complex metabolic changes resulting in increased protein folding capacity, and less dependency on the proteasome. In this study, we show that aminopeptidase B, encoded by the RNPEP gene, is upregulated in bortezomib-resistant myeloma cell lines, and in a murine in vivo model. Moreover, increased RNPEP expression is associated with shorter survival in multiple myeloma patients previously treated with bortezomib-containing regimens. Additionally, expression is increased in plasma cell precursors, a B-lymphoid compartment previously associated with myeloma stem cells. We hypothesized that increased aminopeptidase B expression in aggressive myeloma clones may be used therapeutically toward elimination of the cells via the use of a novel peptide-drug conjugate, melphalan flufenamide (melflufen). Melflufen, a substrate of aminopeptidase B, efficiently eliminates bortezomib-resistant myeloma cells in vitro and in vivo, and completely suppresses clonogenic myeloma growth in vitro at subphysiological concentrations. Thus, melflufen represents a novel treatment option that is able to eradicate drug-resistant myeloma clones characterized by elevated aminopeptidase B expression.

High Immunoproteasome Activity and sXBP1 in Pediatric Precursor B-ALL Predicts Sensitivity towards Proteasome Inhibitors.

Proteasome inhibitors (PIs) are approved backbone treatments in multiple myeloma. More recently, inhibition of proteasome activity with the PI bortezomib has been clinically evaluated as a novel treatment strategy in pediatric acute lymphoblastic leukemia (ALL). However, we lack a marker that could identify ALL patients responding to PI-based therapy. By using a set of activity-based proteasome probes in conjunction with cytotoxicity assays, we show that B-cell precursor ALL (BCP-ALL), in contrast to T-ALL, demonstrates an increased activity of immunoproteasome over constitutive proteasome, which correlates with high ex vivo sensitivity to the PIs bortezomib and ixazomib. The novel selective PI LU015i-targeting immunoproteasome ß5i induces cytotoxicity in BCP-ALL containing high ß5i activity, confirming immunoproteasome activity as a novel therapeutic target in BCP-ALL. At the same time, cotreatment with ß2-selective proteasome inhibitors can sensitize T-ALL to currently available PIs, as well as to ß5i selective PI. In addition, levels of total and spliced forms of XBP1 differ between BCP-ALL and T-ALL, and only in BCP-ALL does high-spliced XBP1 correlate with sensitivity to bortezomib. Thus, in BCP-ALL, high immunoproteasome activity may serve as a predictive marker for PI-based treatment options, potentially combined with XBP1 analyses.

Heterogeneous modulation of Bcl-2 family members and drug efflux mediate MCL-1 inhibitor resistance in multiple myeloma.

Antiapoptotic Bcl-2 family members have recently (re)emerged as key drug targets in cancer, with a tissue- and tumor-specific activity profile of available BH3 mimetics. In multiple myeloma, MCL-1 has been described as a major gatekeeper of apoptosis. This discovery has led to the rapid establishment of clinical trials evaluating the impact of various MCL-1 inhibitors. However, our understanding about the clinical impact and optimal use of MCL-1 inhibitors is still limited. We therefore explored mechanisms of acquired MCL-1 inhibitor resistance and optimization strategies in myeloma. Our findings indicated heterogeneous paths to resistance involving baseline Bcl-2 family alterations of proapoptotic (BAK, BAX, and BIM) and antiapoptotic (Bcl-2 and MCL-1) proteins. These manifestations depend on the BH3 profile of parental cells that guide the enhanced formation of Bcl-2:BIM and/or the dynamic (ie, treatment-induced) formation of Bcl-xL:BIM and Bcl-xL:BAK complexes. Accordingly, an unbiased high-throughput drug-screening approach (n = 528) indicated alternative BH3 mimetics as top combination partners for MCL-1 inhibitors in sensitive and resistant cells (Bcl-xL>Bcl-2 inhibition), whereas established drug classes were mainly antagonistic (eg, antimitotic agents). We also revealed reduced activity of MCL-1 inhibitors in the presence of stromal support as a drug-class effect that was overcome by concurrent Bcl-xL or Bcl-2 inhibition. Finally, we demonstrated heterogeneous Bcl-2 family deregulation and MCL-1 inhibitor cross-resistance in carfilzomib-resistant cells, a phenomenon linked to the MDR1-driven drug efflux of MCL-1 inhibitors. The implications of our findings for clinical practice emphasize the need for patient-adapted treatment protocols, with the tracking of tumor- and/or clone-specific adaptations in response to MCL-1 inhibition.

Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma.

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.