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In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K–PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

NF-κB activation has been linked to prostate cancer progression and is commonly observed in castrate-resistant disease. It has been suggested that NF-κB–driven resistance to androgen-deprivation therapy (ADT) in prostate cancer cells may be mediated by aberrant androgen receptor (AR) activation and AR splice variant production. Preventing resistance to ADT may therefore be achieved by using NF-κB inhibitors. However, low oral bioavailability and high toxicity of NF-κB inhibitors is a major challenge for clinical translation. Dimethylaminoparthenolide (DMAPT) is an oral NF-κB inhibitor in clinical development and has already shown favorable pharmacokinetic and pharmacodyanamic data in patients with heme malignancies, including decrease of NF-κB in circulating leuchemic blasts. Here, we report that activation of NF-κB/p65 by castration in mouse and human prostate cancer models resulted in a significant increase in AR variant-7 (AR-V7) expression and modest upregulation of AR. In vivo castration of VCaP-CR tumors resulted in significant upregulation of phosphorylated-p65 and AR-V7, which was attenuated by combination with DMAPT and DMAPT increased the efficacy of AR inhibition. We further demonstrate that the effects of DMAPT-sensitizing prostate cancer cells to castration were dependent on the ability of DMAPT to inhibit phosphorylated-p65 function.Implications:Our study shows that DMAPT, an oral NF-κB inhibitor in clinical development, inhibits phosphorylated-p65 upregulation of AR-V7 and delays prostate cancer castration resistance. This provides rationale for the development of DMAPT as a novel therapeutic strategy to increase durable response in patients receiving AR-targeted therapy.

Supplementary Figure Legends

Supplementary Figure 7. INPP4B loss does not produce any morphological or cytoskeletal differences.

PDF - 14407K, Supporting histology for Olaparib treatment in vivo.

Supplementary Figure 3. INPP4B's promoter is not directly methylated.

Increased myeloid CD86-expressing APCs following cabozantinib treatment.

Representative images of cell death staining in 22Rv1 empty vector control and p65 overexpressing tumoroids treated +/- DMAPT and/or enzalutamide.

Dexamethasone attenuates the tumor clearance elicited by cabozantinib.

List of qPCR primers.

Significant Tumor Shrinkage following cabozantinib treatment.

Increased CXCL12 within tumor cells by RNA in situ hybridization following cabozantinib treatment.

PDF - 632K, Preclinical assessment of Olaparib and BKM120 treatment in vivo.

Blockade of neutrophil chemotaxis/infiltration rescues cabozantinib-induced tumor regression.

PDF - 546K, Olaparib induces a differing response in vitro depending on the genetic background.

PDF - 2951K, Olaparib induces a differing response in vivo depending on the genetic background.

Cabozantinib-mediated granulocytic infiltration within tumors.

Need as per reviewer suggestion

Supplementary Figure 2. Validation of INPP4B antibody.

Supplementary Figure 1. Generation of Pten+/-Inpp4b+/- and Pten+/-Inpp4b-/- mice.

PDF - 114k, Supporting western blot analysis and drug treatment in prostate cancer cell lines.

PDF - 116K, Supplementary Materials and Methods and Supplementary Figure Legends.

Figure S1. Drug dose-dependent enhancement of γ-H2AX and Rad51 expression using NanoOlaparib; Figure S2. Representative confocal microscopy images of Rad51 and Polθ immunostaining 30 minutes following irradiation.

Supplementary Figure 4. Loss of Inpp4b in MEFs lead to increased Akt activation.

Supplementary Figure Legends

Supplementary Figure 5. Loss of INPP4B results in increased AKT2 activation.

Cabozantinib-mediated tumor clearance is independent of NK and T cells.

PDF - 138K, Survival curves of the in vivo treatment of Olaparib and BKM120.

Supplementary Figures 1-4 from Differential Expression of S6K2 Dictates Tissue-Specific Requirement for S6K1 in Mediating Aberrant mTORC1 Signaling and Tumorigenesis

A major barrier to the successful application of nanotechnology for cancer treatment is the suboptimal delivery of therapeutic payloads to metastatic tumor deposits. We previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anticancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of BSA-coated polymeric nanoparticles (BSA-NPs) into murine PTEN/p53-deficient prostate tumors. On the basis of the observation that BSA coating of NPs enhanced association and internalization by activated neutrophils by approximately 6-fold in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pretreated with cabozantinib. Flow cytometric analysis revealed an approximately 4-fold increase of neutrophil-associated BSA-NPs and an approximately 32-fold increase in mean fluorescent dye uptake following 3 days of cabozantinib/BSA-NP administration, relative to BSA-NP alone. Strikingly, neutrophil depletion with Ly6G antibody abolished dye-loaded BSA-NP accumulation within tumors to baseline levels, demonstrating targeted neutrophil-mediated intratumoral NP delivery. Furthermore, we observed an approximately 13-fold decrease in accumulation of BSA-NPs in the liver, relative to uncoated NPs, post-cabozantinib treatment, suggesting that BSA coating of NPs can significantly enhance cabozantinib-induced, neutrophil-mediated targeted intratumoral drug delivery, while mitigating off-target toxicity. Collectively, we demonstrate a novel targeted nano-immunotherapeutic strategy for enhanced intratumoral delivery of BSA-NPs, with translational potential to significantly augment therapeutic indices of cancer medicines, thereby overcoming current pharmacologic barriers commonly encountered in preclinical/early-phase drug development.

Loss of the tumor suppressor gene Pten in murine prostate recapitulates human carcinogenesis and causes stromal proliferation surrounding murine prostate intraepithelial neoplasia (mPIN), which is reactive to microinvasion. In turn, invasion has been shown to be regulated in part by de novo fatty acid synthesis in prostate cancer. We therefore investigated the effects of genetic ablation of Fasn on invasive potential in prostate‐specific Pten knockout mice. Combined genetic ablation of Fasn and Pten reduced the weight and volume of all the prostate lobes when compared to single knockouts. The stromal reaction to microinvasion and the cell proliferation that typically occurs in Pten knockout were largely abolished by Fasn knockout. To verify that Fasn knockout indeed results in decreased invasive potential, we show that genetic ablation and pharmacologic inhibition of FASN in prostate cancer cells significantly inhibit cellular motility and invasion. Finally, combined loss of PTEN with FASN overexpression was associated with lethality as assessed in 660 prostate cancer patients with 14.2 years of median follow‐up. Taken together, these findings show that de novo lipogenesis contributes to the aggressive phenotype induced by Pten loss in murine prostate and targeting Fasn may reduce the invasive potential of prostate cancer driven by Pten loss. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pervasive event in tumorigenesis due to PI3K mutation and dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Pharmacological inhibition of PI3K has resulted in variable clinical outcomes, however, raising questions regarding the possible mechanisms of unresponsiveness and resistance to treatment. WWP1 is an oncogenic HECT-type ubiquitin E3 ligase frequently amplified and mutated in multiple cancers, as well as in the germ lines of patients predisposed to cancer, and was recently found to activate PI3K signaling through PTEN inactivation. Here, we demonstrate that PTEN dissociated from the plasma membrane upon treatment with PI3K inhibitors through WWP1 activation, whereas WWP1 genetic or pharmacological inhibition restored PTEN membrane localization, synergizing with PI3K inhibitors to suppress tumor growth both in vitro and in vivo. Furthermore, we demonstrate that WWP1 inhibition attenuated hyperglycemia and the consequent insulin feedback, which is a major tumor-promoting side effect of PI3K inhibitors. Mechanistically, we found that AMPKα2 was ubiquitinated and, in turn, inhibited in its activatory phosphorylation by WWP1, whereas WWP1 inhibition facilitated AMPKα2 activity in the muscle to compensate for the reduction in glucose uptake observed upon PI3K inhibition. Thus, our identification of the cell-autonomous and systemic roles of WWP1 inhibition expands the therapeutic potential of PI3K inhibitors and reveals new avenues of combination cancer therapy.

Oncogene-addicted cancers are predominantly driven by specific oncogenic pathways and display initial exquisite sensitivity to designer therapies, but eventually become refractory to treatments. Clear understanding of lung tumorigenic mechanisms is essential for improved therapies. Methods: Lysosomes were analyzed in EGFR-WT and mutant cells and corresponding patient samples using immunofluorescence or immunohistochemistry and immunoblotting. Microtubule organization and dynamics were studied using immunofluorescence analyses. Also, we have validated our findings in a transgenic mouse model that contain EGFR-TKI resistant mutations. Results: We herein describe a novel mechanism that a mutated kinase disrupts the microtubule organization and results in a defective endosomal/lysosomal pathway. This prevents the efficient degradation of phosphorylated proteins that become trapped within the endosomes and continue to signal, therefore amplifying downstream proliferative and survival pathways. Phenotypically, a distinctive subcellular appearance of LAMP1 secondary to microtubule dysfunction in cells expressing EGFR kinase mutants is seen, and this may have potential diagnostic applications for the detection of such mutants. We demonstrate that lysosomal-inhibitors re-sensitize resistant cells to EGFR tyrosine-kinase inhibitors (TKIs). Identifying the endosome-lysosome pathway and microtubule dysfunction as a mechanism of resistance allows to pharmacologically intervene on this pathway. Conclusions: We find that the combination of microtubule stabilizing agent and lysosome inhibitor could reduce the tumor progression in EGFR TKI resistant mouse models of lung cancer.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis

The NPM1 gene is frequently a target of genetic alteration in hematological tumors, particularly of the myeloid lineage. Complete inactivation of Npm1 in the mouse disrupts primitive hematopoiesis and results in embryonic lethality. Npm1 heterozygosity produces features similar to those of MDS that progress to overt leukemia, and specific point mutations of Npm1 lead to bone marrow failure due to loss of hematopoietic stem cells. However, little is known about NPM1’s role in mature, differentiated cells. Here we generated a conditional mouse mutant to inactivate Npm1 across the myelomonocytic lineage, and investigated its ability to influence macrophage maturation and function. We found that Npm1 is not required to maintain macrophage viability, while its loss in mature macrophages reduces production of reactive oxygen species, chemotactic properties and phagocytic capacity. Taking advantage of our recently established Npm1D180del mouse model of ribosome dysfunction and hematological disease, we identify cellular translation and rRNA 2’-O-methlyation as a crucial element in controlling macrophage function. These analyses demonstrate a role for Npm1 in adult immune cells, and reveal the importance of translation regulation in macrophage function.Statement of significanceMacrophages are a major component of the immune response to various insults including to cancer. Here we show that NPM1, the most frequently mutated gene in acute myeloid leukemia, displays a critical role in macrophage function, and we identify ribosome deregulation as one of the underlying mechanisms.

A major barrier to the successful application of nanotechnology for cancer treatment is the efficient delivery of therapeutic payloads to metastatic tumor deposits. We have previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anti-cancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of bovine serum albumin (BSA)-coated polymeric nanoparticles (NPs) into murine PTEN/p53-deficient prostate tumors. Based on the observation that BSA-coating of NPs enhanced association and internalization by activated neutrophils in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pre-treated with cabozantinib. Flow cytometric analysis revealed a 4-fold increase of neutrophil-associated NPs within the tumor microenvironment (TME) of mice pre-treated with cabozantinib relative to untreated controls. At steady-state, following 3 days of cabozantinib/NP administration, 1% of systemically injected dye-loaded NPs selectively accumulated within the TME of mice that were pre-treated with cabozantinib, compared to 0.11% uptake for mice that did not receive cabozantinib pre-treatment. Strikingly, neutrophil depletion with Ly6G antibody abolished NP accumulation in tumors to baseline levels, demonstrating targeted neutrophil-mediated NP delivery to the prostate TME. In summary, we have discovered a novel nano-immunotherapeutic strategy for enhanced intratumoral delivery of injected NPs, which results in significantly higher NP accumulation than reported strategies in the nanotechnology literature to-date.

Purpose: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. Experimental Design: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. Results: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFβ1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFβ1 expression evoke gene-regulatory networks and pathways (TGFβ signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand–VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFβ1 axis reduces tumor cell growth and overcomes drug resistance. Conclusions: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFβ1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Background: CAR-T cells are a tremendous breakthrough in the treatment of certain type of blood cancer, demonstrating impressive and durable responses. However, mechanisms of resistance and relapse have been reported. Mechanisms contributing to relapse after CAR-T therapy include the downregulation of the CAR-T target antigen and the rapid extinguishing of CAR-T cells. We have developed a personalized cancer vaccine whereby patient derived tumor cells are fused to autologous dendritic cells (DC). DC/tumor vaccine induces a broad anti-tumor immunity capable of preventing relapse but may not be effective in patients with advanced disease. Aims: We sought to overcome relapse and resistance to CAR-T therapy to improve the current response rate to CAR-T cells. To this end, we combined CAR-T cells treatment with our personalized DC/tumor vaccine. We postulated that the DC/tumor vaccine would demonstrate synergy with CAR-T cells in a setting where CAR-T cells reduce the bulky disease and the fusion vaccine prevents relapse by expanding CAR-T cells and tumor antigen specific lymphocytes. Methods: To investigate the effects of the CAR-T/fusion vaccine combination, we used the A20 murine B-cell lymphoma model. Syngeneic T cells were obtained from BALB/c mice and retrovirally transduced with a second-generation CAR construct composed of an antigen binding domain that recognizes murine CD19, the CD3ζ domain, 4-1BB as costimulatory molecule and GFP (m19BBz-GFP). GFP expression was used to assess gene-transfer efficiency and monitor the CAR-transduced T cells. The DC/tumor vaccine was obtained by PEG-mediated fusions of A20 cells and BALB/c DC. In the in vitro experiments, T cells transduced with the m19BBz-GFP CAR or non-transduced T cells were co-cultured in the presence or the absence of the DC/A20 vaccine for 3 days. Tumor killing was measured by quantifying Firefly luciferase activity (WT A20) or Renilla luciferase activity (CD19- A20). Vaccine was removed before starting the killing assay. In the in vivo experiment, B-cell lymphoma was induced in BALB/c mice by tail vein injection of A20 cells. The mice were lymphodepleted and treated with m19BBz-GFP CAR-T. The mice were then subcutaneous injected with the DC/A20 fusion vaccine or with PBS (control group). CD8+ T-cells specific for the A20 idiotype epitope were quantified by MHC Class I tetramer analysis. Results: In vitro co-culture of CAR-T cells and DC/A20 vaccine induced a memory-like CAR-T phenotype and strongly improved CAR-T persistence (measured as % of GFP+ T cells in culture). These results were confirmed in vivo where increased CAR-T percentage was observed in the bone marrow and spleen of vaccinated mice with respect to the unvaccinated control group. Moreover, in the vaccinated mice we detected CD8+ T-cells specific for the A20 idiotype epitope demonstrating the expansion of tumor-specific lymphocytes in response to the fusion vaccine. To assess whether the vaccine-induced persistence of CAR-T cells was translated in a higher tumor killing capacity, we performed an in vitro killing assay. To mimic the presence of CD19- clones in the tumor bulk, we used a mixture of WT A20 and CD19-A20 as CAR-T target cells. Enhanced killing capacity against CD19+ tumors was induced by education of the CAR-T with the vaccine. No effects on the CAR-T killing capacity against CD19- A20 were elicited by the fusion vaccine. However, when the same killing assay was performed using as effector cells a mixture of CAR-T and naïve T cells or a mixture of CAR-T and vaccine-educated T cells, we observed that the addition of vaccine-educated T cell to the CAR-T, strongly reduced both A20 WT and A20 CD19- in the cultures. Thus, vaccine-educated T cells are able to kill tumor cells independently from the presence or the absence of the CAR-T target antigen on the tumor. Conclusions: The combination of CAR-T and DC/tumor vaccine, increasing the persistence of CAR-T cells and evoking a polyclonal T cell response against tumor antigens in the non-CAR-transduced T cells, may represent a novel therapeutic strategy to overcame therapeutic resistance and improve current response rate to CAR-T therapy. Disclosures Capelletti: Caris Life Sciences: Current Employment. Stroopinsky: The Blackstone Group: Consultancy. Kufe: REATA: Consultancy, Current equity holder in publicly-traded company; Genus Oncology: Current equity holder in publicly-traded company; Hillstream BioPharma: Current equity holder in publicly-traded company; Canbas: Consultancy. Themeli: Fate Therapeutics: Patents & Royalties. Rosenblatt: Attivare Therapeutics: Consultancy; Imaging Endpoints: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding. Sadelain: Minerva Biotechnologies: Patents & Royalties; Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; Ceramedix: Patents & Royalties; NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); St. Jude Children's Research Hospital: Other: Provision of Services; Atara Biotherapeutics: Patents & Royalties. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

The use of PARP inhibitors in combination with radiotherapy is a promising strategy to locally enhance DNA damage in tumors. Here we show that radiation-resistant cells and tumors derived from a Pten/Trp53-deficient mouse model of advanced prostate cancer are rendered radiation sensitive following treatment with NanoOlaparib, a lipid-based injectable nanoformulation of olaparib. This enhancement in radiosensitivity is accompanied by radiation dose-dependent changes in γ-H2AX expression and is specific to NanoOlaparib alone. In animals, twice-weekly intravenous administration of NanoOlaparib results in significant tumor growth inhibition, whereas previous studies of oral olaparib as monotherapy have shown no therapeutic efficacy. When NanoOlaparib is administered prior to radiation, a single dose of radiation is sufficient to triple the median mouse survival time compared to radiation only controls. Half of mice treated with NanoOlaparib + radiation achieved a complete response over the 13-week study duration. Using ferumoxytol as a surrogate nanoparticle, MRI studies revealed that NanoOlaparib enhances the intratumoral accumulation of systemically administered nanoparticles. NanoOlaparib-treated tumors showed up to 19-fold higher nanoparticle accumulation compared to untreated and radiation-only controls, suggesting that the in vivo efficacy of NanoOlaparib may be potentiated by its ability to enhance its own accumulation. Together, these data suggest that NanoOlaparib may be a promising new strategy for enhancing the radiosensitivity of radiation-resistant tumors lacking BRCA mutations, such as those with PTEN and TP53 deletions. Mol Cancer Ther; 16(7); 1279–89. ©2017 AACR.

Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for functional circRNAs. To address this issue, we leveraged the CRISPR/Cas13d system2 for circRNAs functional screenings. We optimized a strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNAs screening yielded a high rate of false positives phenotypes, while optimized CRISPR/Cas13d led to the identification of bona-fide functional circRNAs. Collectively, we developed a specific and reliable approach to functionalize circRNAs in a high-throughput manner.