In cancer, all roads lead to NADPH.

Cancer cells require increased levels of NADPH for increased nucleotide synthesis and for protection from ROS. Recent studies show that increased NADPH is generated in several ways. Activated AKT phosphorylates NAD kinase (NADK), increasing its activity. NADP formed, is rapidly converted to NADPH by glucose 6-phosphate dehydrogenase and malic enzymes, overexpressed in tumor cells with mutant p53. Calmodulin, overexpressed in some cancers, also increases NADK activity. Also, in IDH1/2 mutant cancer, NADPH serves as the cofactor to generate D-2 hydroxyglutarate, an oncometabolite. The requirement of cancer cells for elevated levels of NADPH provides an opportunity to target its synthesis for cancer treatment.

Anti-Tumor Effects of a Penetratin Peptide Targeting Transcription of E2F-1, 2 and 3a Is Enhanced When Used in Combination with Pemetrexed or Cisplatin.

BACKGROUND: We tested the antitumor effects of a modified E2F peptide substituting D-Arg for L-Arg, conjugated to penetratin (PEP) against solid tumor cell lines and the CCRF-leukemia cell line, alone and in combination with pemetrexed or with cisplatin. For in-vivo studies, the peptide was encapsulated in PEGylated liposomes (PL-PEP) to increase half-life and stability. METHODS: Prostate cancer (DU145 and PC3), breast cancer (MCF7, MDA-MB-468, and 4T1), lymphoma (CCRF-CEM), and non-small cell lung cancer (NSCLC) cell lines (H2009, H441, H1975, and H2228) were treated with D-Arg PEP in combination with cisplatin or pemetrexed. Western blot analysis was performed on the NSCLC for E2F-1, pRb, thymidylate synthase, and thymidine kinase. The H2009 cell line was selected for an in-vivo study. RESULTS: When the PEP was combined with cisplatin and tested against solid tumor cell lines and the CCRF-CEM leukemia cell line, there was a modest synergistic effect. A marked synergistic effect was seen when the combination of pemetrexed and the PEP was tested against the adenocarcinoma lung cancer cell lines. The addition of the PEP to pemetrexed enhanced the antitumor effects of pemetrexed in a xenograft of the H2009 in mice. CONCLUSIONS: The D-Arg PEP in combination with cisplatin caused synergistic cell kill against prostate, breast, lung cancers, and the CCRF-CEM cell line. Marked synergy resulted when the D-Arg PEP was used in combination with pemetrexed against the lung adenocarcinoma cell lines. A xenograft study using the PL-PEP in combination with pemetrexed showed enhanced anti-tumor effects compared to each drug alone.

NAD- and NADPH-Contributing Enzymes as Therapeutic Targets in Cancer: An Overview.

Actively proliferating cancer cells require sufficient amount of NADH and NADPH for biogenesis and to protect cells from the detrimental effect of reactive oxygen species. As both normal and cancer cells share the same NAD biosynthetic and metabolic pathways, selectively lowering levels of NAD(H) and NADPH would be a promising strategy for cancer treatment. Targeting nicotinamide phosphoribosyltransferase (NAMPT), a rate limiting enzyme of the NAD salvage pathway, affects the NAD and NADPH pool. Similarly, lowering NADPH by mutant isocitrate dehydrogenase 1/2 (IDH1/2) which produces D-2-hydroxyglutarate (D-2HG), an oncometabolite that downregulates nicotinate phosphoribosyltransferase (NAPRT) via hypermethylation on the promoter region, results in epigenetic regulation. NADPH is used to generate D-2HG, and is also needed to protect dihydrofolate reductase, the target for methotrexate, from degradation. NAD and NADPH pools in various cancer types are regulated by several metabolic enzymes, including methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, and aldehyde dehydrogenase. Thus, targeting NAD and NADPH synthesis under special circumstances is a novel approach to treat some cancers. This article provides the rationale for targeting the key enzymes that maintain the NAD/NADPH pool, and reviews preclinical studies of targeting these enzymes in cancers.

Rapid Assessment of Surface Markers on Cancer Cells Using Immuno-Magnetic Separation and Multi-frequency Impedance Cytometry for Targeted Therapy.

The rapid qualitative assessment of surface markers on cancer cells can allow for point-of-care prediction of patient response to various cancer drugs. Preclinical studies targeting cells with an antibody to "activated" matriptase conjugated to a potent toxin show promise as a selective treatment for a variety of solid tumors. In this paper, we implemented a novel technique for electrical detection of proteins on surfaces of cancer cells using multi-frequency microfluidic impedance cytometry. The biosensor, consists of two gold microelectrodes on a glass substrate embedded in a PDMS microfluidic channel, is used in conjugation with immuno-magnetic separation of cancer cells, and is capable of differentiating between bare magnetic beads, cancer cells and bead-cell aggregates based on their various impedance and frequency responses. We demonstrated proof-of-concept based on detection of "activated" matriptase proteins on the surface of cultured Mantle cells.

Toward point-of-care assessment of patient response: a portable tool for rapidly assessing cancer drug efficacy using multifrequency impedance cytometry and supervised machine learning.

We present a novel method to rapidly assess drug efficacy in targeted cancer therapy, where antineoplastic agents are conjugated to antibodies targeting surface markers on tumor cells. We have fabricated and characterized a device capable of rapidly assessing tumor cell sensitivity to drugs using multifrequency impedance spectroscopy in combination with supervised machine learning for enhanced classification accuracy. Currently commercially available devices for the automated analysis of cell viability are based on staining, which fundamentally limits the subsequent characterization of these cells as well as downstream molecular analysis. Our approach requires as little as 20 µL of volume and avoids staining allowing for further downstream molecular analysis. To the best of our knowledge, this manuscript presents the first comprehensive attempt to using high-dimensional data and supervised machine learning, particularly phase change spectra obtained from multi-frequency impedance cytometry as features for the support vector machine classifier, to assess viability of cells without staining or labelling.

A phase I trial of MK-2206 and hydroxychloroquine in patients with advanced solid tumors.

PURPOSE: Given the evidence that coordinate inhibition of AKT induces autophagy, we studied the combination of the AKT inhibitor, MK-2206 with hydroxychloroquine (HCQ) in patients with advanced solid tumors. METHODS: Patients were treated with weekly MK-2206 (135 mg or 200 mg) plus HCQ (200 mg, 400 mg or 600 mg BID). RESULTS: Thirty-five patients were enrolled across 5 dose levels. Two DLTs of grade 3 maculo-papular rash were observed at dose level 2 (MK-2206 200 mg weekly plus HCQ at 400 mg BID) and 1 DLT of grade 3 fatigue at dose level 2B (MK-2206 135 mg weekly plus HCQ 600 mg BID). The maximum tolerated dose (MTD) was declared as dose level 2B. The most common adverse events attributed to MK-2206 were hyperglycemia (N = 18; 51%), fatigue (N = 17; 49%), maculo-papular rash (N = 16; 46%), diarrhea (N = 12; 34%), anorexia (N = 11; 31%), and nausea (N = 11; 31%). Patients experiencing adverse events attributed to HCQ were small in number (N = 13) and primarily included fatigue (N = 5; 14%) and maculo-papular rashes (N = 3; 9%). Statistically significant effects on the pharmacokinetic properties of MK-2206 were observed in combination with HCQ. In addition, the plasma concentrations of HCQ in the combination with MK-2206 were significantly higher than the plasma levels of HCQ as monotherapy in prior studies. The best overall response of stable disease was observed in 5/34 (15%) patients. CONCLUSION: The combination of MK-2206 and hydroxychloroquine was tolerable, but with substantial number of drug-related AEs and minimal evidence of antitumor activity.

Safety and enhanced immunostimulatory activity of the DRD2 antagonist ONC201 in advanced solid tumor patients with weekly oral administration.

BACKGROUND: ONC201 is a small molecule antagonist of DRD2, a G protein-coupled receptor overexpressed in several malignancies, that has prolonged antitumor efficacy and immunomodulatory properties in preclinical models. The first-in-human trial of ONC201 previously established a recommended phase II dose (RP2D) of 625 mg once every three weeks. Here, we report the results of a phase I study that evaluated the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of weekly ONC201. METHODS: Patients ≥ 18 years old with an advanced solid tumor refractory to standard treatment were enrolled. Dose escalation proceeded with a 3 + 3 design from 375 mg to 625 mg of ONC201. One cycle, also the dose-limiting toxicity (DLT) window, was 21 days. The primary endpoint was to determine the RP2D of weekly ONC201, which was confirmed in an 11-patient dose expansion cohort. RESULTS: Twenty patients were enrolled: three at 375 mg and 17 at 625 mg of ONC201. The RP2D was defined as 625 mg with no DLT, treatment discontinuation, or dose modifications due to drug-related toxicity. PK profiles were consistent with every-three-week dosing and similar between the first and fourth dose. Serum prolactin and caspase-cleaved cytokeratin-18 induction were detected, along with intratumoral integrated stress response activation and infiltration of granzyme B+ Natural Killer cells. Induction of immune cytokines and effectors was higher in patients who received ONC201 once weekly versus once every three weeks. Stable disease of > 6 months was observed in several prostate and endometrial cancer patients. CONCLUSIONS: Weekly, oral ONC201 is well-tolerated and results in enhanced immunostimulatory activity that warrants further investigation. TRIAL REGISTRATION: NCT02250781 (Oral ONC201 in Treating Patients With Advanced Solid Tumors), NCT02324621 (Continuation of Oral ONC201 in Treating Patients With Advanced Solid Tumors).

A Novel Antibody-Toxin Conjugate to Treat Mantle Cell Lymphoma.

Matriptase is a transmembrane serine protease, synthesized as an inactive single-chain zymogen on the endoplasmic reticulum and transported to the plasma membrane. Matriptase is activated in different epithelial and some B-cell malignancies and changes its conformation and activity is inhibited mainly by its endogenous inhibitor HAI-1. Activated matriptase plays a key role in tumor initiation as well as tumor progression, including invasiveness, and metastasis. To target the anti-mitotic toxin (monomethyl auristatin-E) to activated matriptase, a novel antibody to activated matriptase was conjugated with this toxin via a valine-citrulline-PABA linker. In a previous study, this antibody-toxin conjugate was found to be effective against triple negative breast cancer cell lines and xenografts, alone, or in combination with cisplatin (1). In this study, we examined the anti-tumor effect of the antibody toxin conjugate (ADC) against activated matriptase positive mantle cell lymphoma cell lines (JeKo-1, Maver, Mino, and Z138). This ADC was cytotoxic to these cell lines with IC50s between 5 and 14 µg/mL. The ADC also showed a dose dependent anti-tumor effect on the JeKo-1 xenograft in mice without toxicity.

Checking in on Lenalidomide in Diffuse Large B Cell Lymphoma.

Lenalidomide has modest single-agent activity comparable with other newer drugs in recurrent diffuse large B cell lymphoma with response rates between 19% and 28%. Retrospective series and 1 prospective study suggest that clinically significant responses were predominantly limited to patients with activated B cell (ABC) lymphoma, a finding in agreement with lenalidomide's potent inhibition of nuclear factor κB, the key driver of ABC lymphomas. Recently completed trials will determine whether the additional use of lenalidomide with R/CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) enhances survival compared with R/CHOP alone and whether this activity is limited to ABC lymphomas. Lenalidomide also appears to have activity in the maintenance setting regardless of cell of origin and might play an important role in patients with recurrent disease who are not transplantation candidates. Similarly, because of the ability of lenalidomide to cross the blood-brain barrier, it needs to be further explored in patients with high risk for central nervous system spread. The results of lenalidomide combination studies with chemotherapy and with checkpoint inhibitors are eagerly awaited.

Methods to Study the Role of Methionine-Restricted Diet and Methioninase in Cancer Growth Control.

Cancer cells require exogenous methionine for survival and therefore methionine restriction is a promising avenue for treatment. The basis for methionine dependence in cancer cells is still not entirely clear. While the lack of the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP) is associated with methionine auxotrophy in cancer cells, there are other causes for tumors to require exogenous methionine. Restricting methionine by diet or by enzyme depletion, alone or in combination with certain chemotherapeutics, is a promising antitumor strategy.

Modeling and antitumor studies of a modified L-penetratin peptide targeting E2F in lung cancer and prostate cancer.

E2F1-3a overexpression due to amplification or to mutation or loss of the retinoblastoma gene, induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more of these activating E2Fs is a recognized target in cancer therapeutics. In previous studies we identified by phage display, a novel 7-mer peptide (PEP) that bound tightly to an immobilized consensus E2F1 promoter sequence, and when conjugated to penetratin to increase its uptake into cells, was cytotoxic to several malignant cell lines and human prostate and small cell lung cancer xenografts. Based on molecular simulation studies that showed that the D-Arg penetratin peptide (D-Arg PEP) secondary structure is more stable than the L-Arg PEP, the L-Arg in the peptide was substituted with D-Arg. In vitro studies confirmed that it was more stable than the L- form and was more cytotoxic as compared to the L-Arg PEP when tested against the human castrate resistant cell line, DU145 and the human lung cancer H196 cell line. When encapsulated in PEGylated liposomes, the D-Arg-PEP potently inhibited growth of the DU145 xenograft in mice. Our findings validate D- Arg PEP, an inhibitor of E2F1and 3a transcription, as an improved second generation drug candidate for targeted molecular therapy of cancers with elevated levels of activated E2F(s).

Activated matriptase as a target to treat breast cancer with a drug conjugate.

The antitumor effects of a novel antibody drug conjugate (ADC) was tested against human solid tumor cell lines and against human triple negative breast cancer (TNBC) xenografts in immunosuppressed mice. The ADC targeting activated matriptase of tumor cells was synthesized by using the potent anti-tubulin toxin, monomethyl auristatin-E linked to the activated matriptase-specific monoclonal antibody (M69) via a lysosomal protease-cleavable dipeptide linker. This ADC was found to be cytotoxic against multiple activated matriptase-positive epithelial carcinoma cell lines in vitro and markedly inhibited growth of triple negative breast cancer xenografts and a primary human TNBC (PDX) in vivo. Overexpression of activated matriptase may be a biomarker for response to this ADC. The ADC had potent anti-tumor activity, while the unconjugated M69 antibody was ineffective in a mouse model study using MDA-MB-231 xenografts in mice. Treatment of a human TNBC (MDA-MB-231) showed potent anti-tumor effects in combination with cisplatin in mice. This ADC alone or in combination with cisplatin has the potential to improve the treatment outcomes of patients with TNBC as well as other tumors overexpressing activated matriptase.

Exploiting methionine restriction for cancer treatment.

Normal cells can synthesize sufficient methionine for growth requirements from homocysteine and 5-methyltetrahydrofolate and vitamin B12. However, many cancer-cell types require exogenous methionine for survival and therefore methionine restriction is a promising avenue for treatment. While the lack of the methionine salvage enzyme methylthioadenosine phosphorylase (MTAP) deficiency is associated with methionine dependence in cancer cells, there are other causes for tumors to require exogenous methionine. In this review we describe studies that show restricting methionine to certain cancers by diet or by enzyme depletion, alone or in combination with certain chemotherapeutics is a promising antitumor strategy. The basis for methionine dependence in tumor cells is also briefly reviewed.

Gamma Secretase Inhibition by BMS-906024 Enhances Efficacy of Paclitaxel in Lung Adenocarcinoma.

Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancers (NSCLC). We characterized the interaction between BMS-906024, a clinically relevant Notch gamma secretase inhibitor, and front-line chemotherapy in preclinical models of NSCLC. Chemosensitivity assays were performed on 14 human NSCLC cell lines. There was significantly greater synergy between BMS-906024 and paclitaxel than BMS-906024 and cisplatin [mean combination index (CI) value, 0.54 and 0.85, respectively, P = 0.01]. On an extended panel of 31 NSCLC cell lines, 25 of which were adenocarcinoma, the synergy between BMS-906024 and paclitaxel was significantly greater in KRAS- and BRAF-wildtype than KRAS- or BRAF-mutant cells (mean CI, 0.43 vs. 0.90, respectively; P = 0.003). Paclitaxel-induced Notch1 activation was associated with synergy between BMS-906024 and paclitaxel in the KRAS- or BRAF-mutant group. Knockdown of mutant KRAS increased the synergy between BMS-906024 and paclitaxel in heterozygous KRAS-mutant cell lines. Among KRAS- or BRAF-mutant NSCLC, there was a significant correlation between synergy and mutant or null TP53 status, as well as between synergy and a low H2O2 pathway signature. Exogenous overexpression of activated Notch1 or Notch3 had no effect on the enhanced sensitivity of NSCLC to paclitaxel by BMS-906024. In vivo studies with cell line- and patient-derived lung adenocarcinoma xenografts confirmed enhanced antitumor activity for BMS-906024 plus paclitaxel versus either drug alone via decreased cell proliferation and increased apoptosis. These results show that BMS-906024 sensitizes NSCLC to paclitaxel and that wild-type KRAS and BRAF status may predict better patient response to the combination therapy. Mol Cancer Ther; 16(12); 2759-69. ©2017 AACR.

Small molecule inhibitors block Gas6-inducible TAM activation and tumorigenicity.

TAM receptors (Tyro-3, Axl, and Mertk) are a family of three homologous type I receptor tyrosine kinases that are implicated in several human malignancies. Overexpression of TAMs and their major ligand Growth arrest-specific factor 6 (Gas6) is associated with more aggressive staging of cancers, poorer predicted patient survival, acquired drug resistance and metastasis. Here we describe small molecule inhibitors (RU-301 and RU-302) that target the extracellular domain of Axl at the interface of the Ig-1 ectodomain of Axl and the Lg-1 of Gas6. These inhibitors effectively block Gas6-inducible Axl receptor activation with low micromolar IC50s in cell-based reporter assays, inhibit Gas6-inducible motility in Axl-expressing cell lines, and suppress H1299 lung cancer tumor growth in a mouse xenograft NOD-SCIDγ model. Furthermore, using homology models and biochemical verifications, we show that RU301 and 302 also inhibit Gas6 inducible activation of Mertk and Tyro3 suggesting they can act as pan-TAM inhibitors that block the interface between the TAM Ig1 ectodomain and the Gas6 Lg domain. Together, these observations establish that small molecules that bind to the interface between TAM Ig1 domain and Gas6 Lg1 domain can inhibit TAM activation, and support the further development of small molecule Gas6-TAM interaction inhibitors as a novel class of cancer therapeutics.

First-in-Human Clinical Trial of Oral ONC201 in Patients with Refractory Solid Tumors.

Purpose: ONC201 is a small-molecule selective antagonist of the G protein-coupled receptor DRD2 that is the founding member of the imipridone class of compounds. A first-in-human phase I study of ONC201 was conducted to determine its recommended phase II dose (RP2D).Experimental Design: This open-label study treated 10 patients during dose escalation with histologically confirmed advanced solid tumors. Patients received ONC201 orally once every 3 weeks, defined as one cycle, at doses from 125 to 625 mg using an accelerated titration design. An additional 18 patients were treated at the RP2D in an expansion phase to collect additional safety, pharmacokinetic, and pharmacodynamic information.Results: No grade >1 drug-related adverse events occurred, and the RP2D was defined as 625 mg. Pharmacokinetic analysis revealed a Cmax of 1.5 to 7.5 µg/mL (∼3.9-19.4 µmol/L), mean half-life of 11.3 hours, and mean AUC of 37.7 h·µg/L. Pharmacodynamic assays demonstrated induction of caspase-cleaved keratin 18 and prolactin as serum biomarkers of apoptosis and DRD2 antagonism, respectively. No objective responses by RECIST were achieved; however, radiographic regression of several individual metastatic lesions was observed along with prolonged stable disease (>9 cycles) in prostate and endometrial cancer patients.Conclusions: ONC201 is a selective DRD2 antagonist that is well tolerated, achieves micromolar plasma concentrations, and is biologically active in advanced cancer patients when orally administered at 625 mg every 3 weeks. Clin Cancer Res; 23(15); 4163-9. ©2017 AACR.

NAD+ Kinase as a Therapeutic Target in Cancer.

NAD+ kinase (NADK) catalyzes the phosphorylation of nicotinamide adenine dinucleotide (NAD+) to nicotinamide adenine dinucleotide phosphate (NADP+) using ATP as the phosphate donor. NADP+ is then reduced to NADPH by dehydrogenases, in particular glucose-6-phosphate dehydrogenase and the malic enzymes. NADPH functions as an important cofactor in a variety of metabolic and biosynthetic pathways. The demand for NADPH is particularly high in proliferating cancer cells, where it acts as a cofactor for the synthesis of nucleotides, proteins, and fatty acids. Moreover, NADPH is essential for the neutralization of the dangerously high levels of reactive oxygen species (ROS) generated by increased metabolic activity. Given its key role in metabolism and regulation of ROS, it is not surprising that several recent studies, including in vitro and in vivo assays of tumor growth and querying of patient samples, have identified NADK as a potential therapeutic target for the treatment of cancer. In this review, we will discuss the experimental evidence justifying further exploration of NADK as a clinically relevant drug target and describe our studies with a lead compound, thionicotinamide, an NADK inhibitor prodrug. Clin Cancer Res; 22(21); 5189-95. ©2016 AACR.

Clinical Actionability of Comprehensive Genomic Profiling for Management of Rare or Refractory Cancers.

BACKGROUND: The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. METHODS: A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. RESULTS: Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0-10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. CONCLUSION: Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. IMPLICATIONS FOR PRACTICE: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents.

BMI-1 Targeting Interferes with Patient-Derived Tumor-Initiating Cell Survival and Tumor Growth in Prostate Cancer.

PURPOSE: Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach. EXPERIMENTAL DESIGN: We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1. RESULTS: We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell-like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor-directed therapies, without toxic effects on normal tissues. CONCLUSIONS: Our data offer a paradigm for targeting TICs and support the development of BMI-1-targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176-91. ©2016 AACR.