CRISPR Dropout Screens Research Articles

Abstract IA019: Identification of metabolic adaptation mechanisms that confer resistance to glutathione depletion in Ewing sarcoma

Abstract Aggressive cancer cells must overcome diverse stress forms in the tumor microenvironment and circulation, such as oxidative stress, to survive and metastasize. One adaptive process to counter oxidative stress is to upregulate glutathione (GSH), a tripeptide consisting of glutamate, cysteine, and glycine, which is a powerful antioxidant in cells, including in tumor cells. A rate-liming step in GSH production is the availability of the amino acid, cysteine. Tumor cells can either import cystine, a dimer of cysteine, through the xCT antiporter system, for conversion to the reduced cysteine monomer, or in some tumor types, they can synthesize cysteine de novo from methionine and serine through the transsulfuration pathway. GSH is not only critical for redox homeostasis, but is taken up into mitochondria where is acts as a source of sulphur to generate iron-sulphur (Fe-S) clusters that are essential for many mitochondrial enzymes including electron transport complexes. We wished to determine if Ewing sarcoma cells could tolerate reduced levels of GSH, using either xCT genetic or pharmacologic inhibition, or by blocking downstream enzymes in the GSH synthesis pathway, including GCLC using buthionine sulfoximine (BSO), or glutaminase using CB-839. To our surprise, we were able to generate BSO and CB-839 resistant cells with vanishingly low levels of GSH. These cells somehow maintained not only redox homeostasis, but also showed functional mitochondrial respiration (oxidative phosphorylation). We then performed CRISPR dropout screens to identify how these cells might be surviving in the absence of GSH, and to tease out specific dependencies of these BSO and CB-839 resistant cells. These studies revealed unexpected dependencies, and opportunities for synthetic lethal interventions, as will be discussed. Citation Format: Poul H. Sorensen, Hai-Feng Zhang, Christopher Hughes, Alberto Delaidelli, Samuel Aparicio. Identification of metabolic adaptation mechanisms that confer resistance to glutathione depletion in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA019.

Integrated multi-omics analyses identify anti-viral host factors and pathways controlling SARS-CoV-2 infection

Host anti-viral factors are essential for controlling SARS-CoV-2 infection but remain largely unknown due to the biases of previous large-scale studies toward pro-viral host factors. To fill in this knowledge gap, we perform a genome-wide CRISPR dropout screen and integrate analyses of the multi-omics data of the CRISPR screen, genome-wide association studies, single-cell RNA-Seq, and host-virus proteins or protein/RNA interactome. This study uncovers many host factors that are currently underappreciated, including the components of V-ATPases, ESCRT, and N-glycosylation pathways that modulate viral entry and/or replication. The cohesin complex is also identified as an anti-viral pathway, suggesting an important role of three-dimensional chromatin organization in mediating host-viral interaction. Furthermore, we discover another anti-viral regulator KLF5, a transcriptional factor involved in sphingolipid metabolism, which is up-regulated, and harbors genetic variations linked to COVID-19 patients with severe symptoms. Anti-viral effects of three identified candidates (DAZAP2/VTA1/KLF5) are confirmed individually. Molecular characterization of DAZAP2/VTA1/KLF5-knockout cells highlights the involvement of genes related to the coagulation system in determining the severity of COVID-19. Together, our results provide further resources for understanding the host anti-viral network during SARS-CoV-2 infection and may help develop new countermeasure strategies.

CRISPR Screen of Druggable Targets in Small Cell Lung Cancer Identified ATM Inhibitor (AZD1390) as a Radiosensitizer

Small cell lung cancer (SCLC) is an aggressive and lethal form of lung cancer and the overall 5-year survival (OS) for patients is a dismal 7%. Radiation therapy (RT) provides some benefit for selected patients with SCLC but could be improved with radiosensitizing agents. In this study, we identified novel radiosensitizers for SCLC by a CRISPR-Cas9 screen and evaluated the efficacy of ATM inhibitor AZD1390 as a radiosensitizer of SCLC. We transduced the SCLC cell line SBC5 with a custom CRISPR sgRNA library focused on druggable gene targets and treated cells with RT. Cells collected at multiple timepoints were subjected to next-generation sequencing. We determined radiosensitization both in vitro with cell lines assessed by short-term viability and clonogenic assays, and in vivo mouse models by tumor growth delay. Pharmacodynamic effects of AZD1390 were quantified by ATM-Ser1981 phosphorylation, and RT-induced DNA damage by comet assay. Using a CRISPR dropout screen, we identified multiple radiosensitizing genes for SCLC at various timepoints with ATM as a top determinant gene for radiosensitivity. Validation by ATM knockout (KO) demonstrated increased radiosensitivity by short-term viability assay (dose modification factor [DMF]50 = 3.25-3.73 in SBC5 ATM-KO) and clonogenic assays (DMF37 1.25-1.65 in SBC5 ATM-KO). ATM inhibition by AZD1390 effectively abrogated ATM Ser1981 phosphorylation in SCLC cell lines and increased RT-induced DNA damage. AZD1390 synergistically increased the radiosensitivity of SCLC cell lines (cell viability assay: SBC5 DMF37 = 2.19, SHP77 DMF37 = 1.56, H446 DMF37 = 3.27, KP1 DMF37 = 1.65 at 100nM; clonogenic assay: SBC5 DMF37 = 4.23, H1048 DMF37 = 1.91), and in vivo murine syngeneic, KP1, and patient-derived xenograft (PDX) models, JHU-LX108 and JHU-LX33. In this study, we demonstrated that genetically and pharmacologically (AZD1390) inhibiting ATM markedly enhanced RT against SCLC, providing a novel pharmacologically tractable radiosensitizing strategy for patients with SCLC.

NPM1c Mutations Are Associated with Reduced Levels of Multiple Ribosome Biogenesis Factors Leading to Therapeutic Vulnerabilities

Objectives: Mutations in the NPM1 gene are found in more than 30% of acute myeloid leukaemia (AML) cases. The mutations disrupt a nucleolar localization signal (NoLS) and create a novel nuclear export signal (NES), leading to cytoplasmic displacement of the protein (NPM1c). NPM1cmutations prime haematopoietic progenitors to leukaemic transformation, but their precise molecular consequences remain elusive. Here, we first examine the effects of isolated NPM1c mutations on mouse haematopoietic cells and explore their impact on their transcriptome, including at single-cell resolution, and their proteome. Then, using several murine and human AML models and datasets we investigate the role of such NPM1c-associated changes in fully transformed AML cells and explore their potential as therapeutic vulnerabilities. Methods: We performed RNA-seq, CITE-seq and TMT global quantitative proteomics using lineage-depleted bone marrow progenitor cells from NPM1 flox-cA/+;Mx1-cre mice ( Vassiliou et al, Nat Genet, 2011). Proteomic changes were contrasted to those identified in a recently published dataset of 44 human AMLs, including 8 NPM1-mutant AMLs. Candidate vulnerabilities were identified using datasets from CRISPR-Cas9 drop-out screens and were validated in NPM1 cA/+-harbouring cell lines and primary mouse NPM1 cA/+;FLT3 ITD/+ AMLs. Results: We found a global increase in the protein abundance of several members of the KPN family in pre-leukaemic Npm1 cA/+ cells, an effect also observed in human NPM1c+ AMLs. Moreover, we observed a decrease in the relative abundance of several ribosome biogenesis-related proteins in pre-leukaemic Npm1 cA/+ cells and confirmed that this effect persists in human NPM1c+ AML patient samples. We go on to show that, when compared to NPM1 +/+ cells, pre-leukaemic NPM1 cA/+cells show high sensitivity to Actinomycin D and other RNA pol I inhibitors, but not to the translation inhibitor cycloheximide. Combination treatment with Actinomycin D and Venetoclax inhibited the growth and colony forming ability of both pre-leukaemic NPM1 cA/+cells and primary mouse Npm1 cA/+;Flt3 ITD/+AMLs. By contrasting our findings with data from CRISPR drop-out screens, including a genome-wide screen in primary mouse Npm1 cA/+;Flt3 ITD/+/Cas9AMLcells, we identified and validated ribosome biogenesis factors, such as Tsr3, whose knock-out selectively affected cell growth, survival and clonogenicity of NPM1 +/+AML cells, therefore representing novel potential therapeutic targets that could be exploited in the clinic. Conclusions: We show that NPM1c mutations are associated with reduced levels in several ribosome biogenesis factors resulting in therapeutic vulnerabilities of NPM1-mutant pre-leukaemic and leukaemic (AML) cells. The development of novel therapeutics to target these factors may help improve the survival of patients with NPM1-mutant AML. Figure Legend: (A) Volcano plot showing protein abundance in mouse NPM1 cA/+vs NPM1 +/+ pre-leukaemic haematopoetic progenitors. (B) Relative abundance of ribosome-related proteins in human NPM1 cA/+vs NPM1 +/+AML samples (dataset from Kramer et al., 2022).

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

The Zymogen Form of Caspase-1 Is Required to Fine Tune Excessive Cell-Intrinsic Inflammation in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is characterized by the expansion of clonally-derived mutant hematopoietic stem and progenitor cells. Many patients do not respond to standard chemotherapy because AML cells acquire mutations that exploit inflammatory and cell death pathways, allowing them to evade cell death. Targeted therapies are urgently needed to block AML cell pro-survival and inflammatory signaling for more effective outcomes. One promising area of interest is Caspase-1 (CASP1), a cysteine-aspartic acid protease responsible for cleaving the precursors of interleukin-1β (IL-1β) and Gasdermin D, which induces pyroptosis, into their active mature peptides. In myeloid malignancies, IL-1β levels are significantly elevated, and pyroptosis has been linked to ineffective hematopoiesis as observed in MDS, thereby implicating CASP1. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme. In this report, we present a novel and unexpected role for Pro-CASP1, which appears to be independent of its proteolytic function, in MDS/AML leukemic cells. Pyroptosis, an inflammatory cell death mechanism, is driven by CASP1-mediated osmotic lysis and IL-1b secretion. Notably, CASP1 expression is significantly higher in AML patients compared to healthy hematopoietic cells, and elevated CASP1 expression in AML patients correlates with shorter overall survival. In leukemic cells, CASP1 primarily exists in its zymogen form, lacking the ability to induce pyroptosis. It is thus unexpected that elevated CASP1 expression, which typically leads to heightened susceptibility to pyroptotic cell death, does not result in pyroptosis in leukemic cells, and the underlying mechanisms for this evasion remain unknown. To investigate Pro-CASP1's significance in AML cells, we used CRISPR-Cas9 to create Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic MDS and AML cell lines (THP1 and MDSL). Pro-CASP1 KO MDS/AML cells showed reduced cell growth and leukemic colony forming potential in methylcellulose compared to isogenic WT control cells. When Pro-CASP1 KO THP1 AML cells were transplanted into immunocompromised mice, the loss of Pro-CASP1 significantly reduced leukemic cell burden and extended overall survival. We next preformed experiments to probe the mechanism of action of Pro-CASP1 in AML cells. Previous studies suggest that Pro-CASP1 can function as a scaffold to elicit downstream signaling without cleaving into its activated form. The functional defect in Pro-CASP1 KO THP1 AML cells was rescued with full-length Pro-CASP1, but not with a scaffolding mutant of CASP1, indicating a specific dependency of leukemic cells on the zymogen form of CASP1. To precisely delineate which domains of Pro-CASP1 are essential for AML, we performed a tiled CRISPR dropout screen in MDSL and THP1 cells. The results showed similar CRISPR-Knockout High Sensitivity (CKHS) domain footprints in both cell lines mapping to the N-terminal CARD and the Inter-Domain Linker (IDL) domains. These results indicate that Pro-CASP1's proteolytic function was found to be dispensable, whereas the CARD domain, a scaffolding region, was crucial for leukemogenesis. We next investigated how loss of Pro-CASP1 affects inflammatory signaling in AML cells. Deletion of Pro-CASP1 in THP1 AML cells led to excessive and constitutive activation of NF-kB as indicated by nuclear translocation and phosphorylation of p65/RelA. Restoring NF-kB regulation using the inhibitor of kappa B (IkB) super-repressor (IkB-SR) was sufficient to reverse the leukemic progenitor defect and cell viability in Pro-CASP1 KO AML cells. These findings suggest that Pro-CASP1 is required for AML cells to finetune canonical NF-kB activation. Conversely, loss of Pro-CASP1 results in excessive NF-kB activation that is incompatible with AML, consistent with previous observations (Ellegast et al., Cancer Discovery 2022). In summary, Pro-CASP1 serves as a critical signaling hub, fine-tuning NF-kB pathways independently of its proteolytic function in leukemic cells. Its loss exposes leukemic cells to functional and therapeutic vulnerabilities.

Identifying Stress Granules As Determinants of Leukemia Stem Cell Maintenance and Stress Adaptation

The development of novel leukemic stem cell (LSC)-targeted therapeutics, which spare hematopoietic stem cells (HSC), is an urgent goal towards reducing the high, LSC-driven, relapse rates in acute myeloid leukemia (AML) patients. Towards elucidating key post-transcriptional networks underlying LSC maintenance we previously performed an in vivo pooled CRISPR dropout screen targeting 128 RNA binding proteins (RBPs) highly expressed in AML LSCs while having low expression in HSCs. This screen highlighted 12 RBPs crucial for murine AML LSC-mediated leukemic growth, of which there was a significant enrichment of stress granule (SG) RBPs. Further, gene set enrichment analysis shows the SG proteome is enriched in human AML LSCs vs non-LSCs while normal HSCs have reduced SG RBP expression relative to multipotent progenitor cells. SGs are dynamic biomolecular condensates, composed of ribonucleoprotein complexes, which may form as a protective mechanism against a variety of stress conditions. SGs are gaining increasing attention as contributors to cancer metastasis and chemoresistance, but their role in LSCs is virtually unexplored. The intriguing enrichment of SG RBPs in our screen hints at a larger role for SGs in LSC maintenance and/or stress resistance. To address this, we focused on the core SG nucleation RBP, G3BP1, a molecular switch that guides SG assembly. Fluorescence microscopy was first performed to describe SG dynamics in leukemia vs primitive normal cells where interestingly, a subset of patient AML cells contained G3BP1 SGs in the absence of added stress. In contrast, primitive healthy murine stem cells had reduced heat shock-induced SG formation in comparison to downstream normal progenitors. A series of shG3BP1 knockdown experiments performed in human leukemia cell lines and patient AML specimens demonstrated that shG3BP1-transduced cells showed increased apoptosis and differentiation and reduced colony forming capacity and competition in vitro compared to control cells. The same set of assays showed minimal impact of SG impairment to healthy umbilical cord blood cells. Importantly, in vivo xenotransplantation demonstrated that LSCs with G3BP1 knocked down were significantly compromised in their capacity to generate leukemic grafts whereas normal HSCs assessed in a similar manner display much less G3BP1 dependency. Towards determination of the SG mechanisms essential for bulk AML and LSC regulation, we performed matched RNA-sequencing and proteomics in G3BP1 knocked down AML cells as well as BioID in AML cell lines, primary patient AML cell subsets, and healthy hematopoietic cells to allow for the uncovering of an AML specific SG proteome. These analyses highlighted numerous potential SG mechanisms of cancer cell stress resistance and/or stem cell regulatory processes including sequestration of apoptotic factors and oncogenic signalling pathways. Of note, RNA-seq and proteomics in SG impaired shG3BP1 leukemia cells showed both a negative enrichment of the SG proteome and LSC signature as well as a positive enrichment of the innate immune response. Interestingly, our BioID uncovered that the AML specific SG proteome is also enriched for positive regulation of RIG-I signaling pathway (GO: Biological Process). Together this data suggests SGs play a protective role through the downregulation of overactive innate immune signalling in AML. Work using optimized small input approaches to map SG RNA and protein interactomes in LSCs vs HSCs to confirm similar regulatory relationships as well as identify LSC-context specific ones is ongoing. Overall, these results elucidate SGs as key AML LSC stress adaptive regulatory networks and highlights them as novel targets for consideration in AML therapeutics.

Temporal chromatin accessibility changes define transcriptional states essential for osteosarcoma metastasis

The metastasis-invasion cascade describes the series of steps required for a cancer cell to successfully spread from its primary tumor and ultimately grow within a secondary organ. Despite metastasis being a dynamic, multistep process, most omics studies to date have focused on comparing primary tumors to the metastatic deposits that define end-stage disease. This static approach means we lack information about the genomic and epigenomic changes that occur during the majority of tumor progression. One particularly understudied phase of tumor progression is metastatic colonization, during which cells must adapt to the new microenvironment of the secondary organ. Through temporal profiling of chromatin accessibility and gene expression in vivo, we identify dynamic changes in the epigenome that occur as osteosarcoma tumors form and grow within the lung microenvironment. Furthermore, we show through paired in vivo and in vitro CRISPR drop-out screens and pharmacological validation that the upstream transcription factors represent a class of metastasis-specific dependency genes. While current models depict lung colonization as a discrete step within the metastatic cascade, our study shows it is a defined trajectory through multiple epigenetic states, revealing new therapeutic opportunities undetectable with standard approaches.

Therapeutic targeting of metabolic vulnerabilities in cancers with MLL3/4-COMPASS epigenetic regulator mutations.

Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity toward increased purine synthesis in MLL3/4-KO mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA-Seq identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4-KO cells. Mechanistically, we demonstrated that compensation by MLL1/COMPASS was underlying these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vitro and in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.

DIPG-45. ELONGIN B REGULATES H3K27M CHROMATIN INCORPORATION TO PROMOTE MALIGNANCY IN DIFFUSE MIDLINE GLIOMA

Abstract Chromatin dysregulation is both a cause and consequence of aberrant RNA Polymerase II (Pol2) transcription in cancer. Some of the most striking examples of chromatin disruption in cancer include somatic mutations in genes encoding histone H3 (H3.3/H3.1K27M) observed in ~80% of diffuse midline glioma (DMG). H3K27M mutant histones act in a dominant manner to disrupt chromatin regulation and Pol2-dependent transcription in DMG. How H3K27M impinges on the Pol2 transcriptional machinery and whether aberrant Pol2 transcription can reciprocally induce chromatin dysregulation in DMG remain open questions. We conducted epigenome-focused CRISPR dropout screens and identified multiple regulators of Pol2 elongation, including Elongin B and C (ELOB/ELOC) as DMG genetic dependencies. Follow-up studies confirm that knockout (KO) of ELOB inhibits DMG proliferation in neurosphere culture and abrogates DMG tumorigenesis in orthotopic xenografts. Additional chromatin profiling studies reveal that ELOB binding sites are enriched in H3K27M mutant histones, suggesting that ELOB works together with H3K27M to promote malignant gene expression in DMG. Indeed, reciprocal knockout studies suggest that while ELOB chromatin binding patterns were not altered by H3K27M KO, ELOB KO significantly reduced H3K27M chromatin incorporation at thousands of genomic loci. Further RNAseq and PROseq analyses reveal that ELOB KO alters Pol2 transcription resulting in the up-regulation of genes associated with neuronal and muscle differentiation and the repression of glial stem cell genes. Together, these findings suggest that H3K27M chromatin binding patterns and aberrant gene regulation in DMG are, in part, dictated by ELOB and the Pol2 elongation machinery. Finally, analyses of patient datasets indicate that ELOB complex genes are overexpressed in high grade glioma and DMG and correlate with decreased patient survival. Collectively, these studies suggest that a functional interaction between ELOB and H3K27M mutant histones induces DMG-specific aberrations in chromatin and gene regulation to drive malignancy.

Abstract IA019: How to target lung metastasis: dynamic changes in chromatin accessibility during osteosarcoma lung metastasis expose vulnerabilities

Abstract The metastatic cascade describes the series of steps required for a cancer cell to successfully spread from its primary tumor to a secondary organ. Despite knowing that metastasis is a fluid process, almost all studies to date have relied on end-point assays. As a result, little is known about the continuum of molecular events required for successful growth within the secondary organ. By profiling accessible chromatin in osteosarcoma cells grown in vitro and isolated from an early and late timepoint during lung metastasis, we identify temporally distinct epigenomic reprogramming events that occur as tumors form and grow within the metastatic microenvironment. Furthermore, these data pinpoint key upstream transcription factors responsible for the dynamic chromatin changes. Through paired in vivo and in vitro CRISPR drop-out screens, we show a subset of these putative pro-metastatic transcription factors are essential for lung metastasis, but dispensable for in vitro growth. Targeting the same factors with chemical probes in vitro and ex vivo demonstrated similar context-dependent essentiality. Altogether, our study demonstrates the epigenomes of metastasizing cancer cells are tightly regulated in a dynamic and context-specific manner, and the transcription factors controlling this process may serve as novel vulnerabilities. Citation Format: Peter C. Scacheri. How to target lung metastasis: dynamic changes in chromatin accessibility during osteosarcoma lung metastasis expose vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA019.

HGG-51. Uncovering therapeutic vulnerabilities in mismatch repair-deficient gliomas

INTRODUCTION: We have observed that approximately 26% of recurrent gliomas acquire hypermutation following treatment with temozolomide (TMZ). Intriguingly, 91% of these tumors harbor mutations in mismatch repair (MMR) genes. Since MMR deficiency confers resistance to TMZ, strategies to target MMR-deficient gliomas stand to impact many patients. METHODS: We ablated the MMR genes MSH2, MSH6, MLH1, and PMS2 using an all-in-one sgRNA-CRISPR/Cas9 expression vector to generate isogenic MMR knockouts in patient-derived glioma cell lines. We characterized the gene expression profiles of these MMR-deficient glioma models and leveraged high-throughput drug screens and genome-scale CRISPR/Cas9 dropout screens to identify therapeutic vulnerabilities induced by loss of MMR. RESULTS: We show that loss of each major MMR gene confers resistance to TMZ. Gene set enrichment analysis of our MMR-deficient knockouts shows enrichment of several hallmark gene sets including DNA repair and G2M checkpoint signatures, and our genome-wide CRISPR dropout screen reveals that MMR-deficient cells are preferentially dependent on a number of genes involved in DNA repair and cell cycle, along with several other pathways. Lastly, the high-throughput drug repurposing (REPO) screen shows that loss of MMR confers differential dependencies to small molecule inhibitors. CONCLUSIONS: Using CRISPR/Cas9 to knock out individual MMR pathway members allows us to systematically study the response of MMR-deficient cells to alkylating agents in an isogenic context. Importantly, these isogenic models reveal that MMR-deficient glioma cells possess novel genetic dependencies and sensitivities to small molecules, which may inform future therapies for MMR-deficient tumors.

Abstract P096: Using CRISPR-Cas9 screens to identify microRNA involved in aggressive prostate cancer phenotypes

Abstract Background: Prostate cancer (PCa) is the most common cancer diagnosis in Canadian men. For many, the tumour can remain “dormant” negating a need for treatment, but if the tumour progresses and becomes metastatic, prognosis is poor. Despite the introduction of new treatments, metastatic PCa remains a lethal and incurable disease. This is invariably due to the onset of castration-resistance in advanced cases producing a more aggressive tumour with increased metastatic potential. We hypothesize that microRNA can promote these aggressive PCa phenotypes due to their ability to regulate diverse gene networks simultaneously. Methods: We have generated miRKOv2, the second version of our microRNA CRISPR Knockout library using the latest on- and off-target scoring algorithms and other microRNA-specific features for CRISPR-Cas9 guide RNA (gRNA) design. miRKOv2 will be used in three separate screens to identify microRNA that are essential for PCa growth, microRNA that are involved in PCa metastasis, and microRNA that can confer castration-resistance, respectively. Results: Next generation sequencing of the plasmid miRKOv2 library showed a median gRNA coverage of ~1100X. In silico comparisons to other microRNA libraries demonstrate that miRKOv2 is a superior sgRNA library. DU145 cells stably expressing Cas9 are highly active and proof of principle growth assays demonstrate that essential gene knockout and subsequent decrease in cell viability is detectable. Generation of a Cas9+ PCa cell line panel and screens are ongoing. Conclusion: miRKOv2 is an improved microRNA-focused CRISPR library with high gRNA coverage. Proof of principle assays demonstrate that knockout of essential genes results in a reduction in cell viability, indicating that a CRISPR dropout screen is appropriate for our in vitro model. This project aims to identify microRNA dependencies in metastatic and castration-resistant PCa that can be leveraged into new biomarkers and therapies. Citation Format: Jonathan Tak-Sum Chow, Daniel K. C. Lee, Martino Marco Gabra, Norman Fu, Keyue Chen, Leonardo Salmena. Using CRISPR-Cas9 screens to identify microRNA involved in aggressive prostate cancer phenotypes [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P096.

CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer

Cancer stem cells (CSCs) are responsible for tumor progression, recurrence, and drug resistance. To identify genetic vulnerabilities of colon cancer, we performed targeted CRISPR dropout screens comprising 657 Drugbank targets and 317 epigenetic regulators on two patient-derived colon CSC-enriched spheroids. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells yielded therapeutic candidates. We unraveled 44 essential genes for colon CSC-enriched spheroids propagation, including key cholesterol biosynthetic genes (HMGCR, FDPS, and GGPS1). Cholesterol biosynthesis was induced in colon cancer tissues, especially CSC-enriched spheroids. The genetic and pharmacological inhibition of HMGCR/FDPS impaired self-renewal capacity and tumorigenic potential of the spheroid models in vitro and in vivo. Mechanistically, HMGCR or FDPS depletion impaired cancer stemness characteristics by activating TGF-β signaling, which in turn downregulated expression of inhibitors of differentiation (ID) proteins, key regulators of cancer stemness. Cholesterol and geranylgeranyl diphosphate (GGPP) rescued the growth inhibitory and signaling effect of HMGCR/FDPS blockade, implying a direct role of these metabolites in modulating stemness. Finally, cholesterol biosynthesis inhibitors and 5-FU demonstrated antitumor synergy in colon CSC-enriched spheroids, tumor organoids, and xenografts. Taken together, our study unravels novel genetic vulnerabilities of colon CSC-enriched spheroids and suggests cholesterol biosynthesis as a potential target in conjunction with traditional chemotherapy for colon cancer treatment.

In This Issue

See article, p. 434.Ficlatuzumab is a first-in-class monoclonal antibody that blocks oncogenic MET signaling by blocking its ligand HGF. Here Wang et al. report on the safety and efficacy of ficlatuzumab combination with cytarabine in 17 patients with relapsed/refractory acute myeloid leukemia. Nine of the patients (53%) achieved a complete remission. Progression-free survival was 31.2 months, and median overall survival has not been reached. These outcomes compare favorably to historical data but remain to be validated in larger, randomized trials. Phosphoproteomic analysis identified S6 and MET phosphorylation as molecular correlates of response. Ras and GTPase gene expression signature was higher in prospective responders, whereas prospective nonresponders had elevated interferon and Myc signature.See article, p. 450.Human T-cell leukemia virus type-1 (HTLV-1) infection can progress to adult T-cell leukemia/lymphoma (ATL). To elucidate determinants of HTLV-1–driven oncogenesis, Koya et al. characterize circulating blood cells of healthy donors, HTLV-1 carriers, and patients with ATL by genome sequencing and CITE-seq combined with TCR-seq and BCR-seq. This high-dimensional single-cell analysis identifies Treg phenotype as a hallmark of premalignant clonal outgrowth. As the neoplastic clones progress to ATL, their expansion is accompanied by a proportional expansion of the myeloid compartment and immunosuppressive phenotypes including PD-L1 upregulation. The study delineates a new mechanism of PD-L1 acquisition by protein transfer from ATL to nonmalignant cells.See article, p. 468.Immunologic consequences of cell death contribute to clinical efficacy of certain chemotherapeutics, and identification of immunogenic drugs is essential for their optimal clinical use alone and in combination with immunotherapy. In this work, Gulla et al. have shown that a specific anti–multiple myeloma (MM) immunity occurs upon cell death induced by the proteasome inhibitor bortezomib, leading to immunologic memory and long-term clinical response. Intrinsic activation of STING followed by a viral mimicry state further contributes to the immunogenic effect, and combination of bortezomib with a STING agonist potentiates tumor cell–immune microenvironment interplay. These findings invite a deeper characterization of the immunologic effects of anti-MM agents and inform novel chemo-immunotherapy combinations.See article, p. 484.To obtain insight into the etiology of childhood leukemia, Brandsma et al. determined mutation burden of leukemic blasts and normal hematopoietic stem and progenitor cells (HSPC) from pediatric acute myeloid leukemia (pAML) patients and healthy age-matched controls. Integrating these data with available adult cohorts, they established a baseline of HSPC mutation accrual over the entire human lifespan. A subset of pediatric leukemia cases falling above the baseline harbored more genetic driver events and associated with better survival. The mutation pattern, characteristic of cells with high proliferation and oxidative stress, suggests a more committed progenitor as a cell of origin in this pAML subset.See article, p. 500.While the most frequent drivers of clonal hematopoiesis have been characterized previously, mutations in these genes are not present in about half of clonal hematopoiesis cases. By identifying recurrent mutations in the blood of large numbers of healthy people, Beauchamp et al. discover additional genes likely to drive clonal hematopoiesis and thus regulating hematopoietic stem cell (HSC) expansion. Modeling ZBTB33 mutations in mouse HSCs, the authors observe that the edited cells expand and have a competitive advantage compared with control cells. Cells with Zbtb33 mutations also have altered intron retention, potentially connecting ZBTB33 to RNA splicing, a commonly mutated pathway in clonal hematopoiesis and myelodysplastic syndromes.See article, p. 518.Romine et al. characterize acute myeloid leukemia (AML) sensitivity to BET inhibitors (BETi) by two orthogonal approaches: ex vivo drug screening of genetically defined primary AML samples, and genome-wide CRISPR screens of BETi-sensitive and BETi-resistant cells. Both identify loss of monocytic differentiation regulators as drivers of BETi resistance. BETi-resistant AML cells express low levels of myeloid markers; conversely, forced myeloid differentiation sensitizes AML cells to BETi. CRISPR dropout screens in BETi-resistant cells reveal BCL2 and CDK4/6 as targetable vulnerabilities to overcome BETi resistance. Moreover, monocytic AML refractory to BCL2 inhibition, an unmet clinical challenge, shows preclinical responses to combined Bcl2 and BETi.See article, p. 532.Binding of tyrosine kinase inhibitors (TKI) by plasma proteins, most notably alpha-1-acid glycoprotein (AGP), can dramatically reduce TKI bioavailability and thus clinical potency. In this work, Young et al. characterize AGP impact on staurosporine-derived TKI (STS-TKI) activity against FLT3-mutated acute myeloid leukemia (AML). Under physiologic conditions and drug concentrations, AGP quenches STS-TKI activity to subtherapeutic levels due to high-affinity drug–protein interactions. To overcome this, the authors screen for drugs restoring TKI activity in the presence of AGP, identifying mifepristone. In an in vivo model, mifepristone coadministration restores midostaurin clearance of AML cells. These results suggest a combinatorial approach for unlocking TKI activity to potentiate therapeutic responses in FLT3-mutated AML.

Abstract 1021: Investigating the mechanism of PARP7 inhibition in type I interferon signaling by arrayed CRISPR screening

Abstract Genomic instability in cancer cells leads to cellular stress through the accumulation of aberrant nucleic acid species in the cytosol. We have shown that PARP7, a monoPARP, is a negative regulator of cytosolic nucleic acid sensing in cancer cells. RBN-2397 is a potent and selective PARP7 inhibitor that induces antitumor immunity in preclinical models and is currently being evaluated in a Phase I clinical trial. In our preclinical investigations, we found that in a subset of cancer cell lines, such as NCI-H1373, inhibition of PARP7 triggers Type I IFN release, STAT1 phosphorylation, and growth arrest. In contrast, other cell lines, for example, HARA, do not mount an IFN-response upon PARP7 inhibition, even though they are responsive to transfection of exogenous nucleic acids and PARP7 is expressed and enzymatically active. To investigate the underlying mechanism of PARP7 inhibition and to determine the drivers of the differential sensitivity across cell lines we performed arrayed CRISPR knockout screens, targeting approximately 240 genes in the nucleic acid sensing and IFN signaling pathways, in the presence and absence of PARP7 inhibition. Our arrayed screens confirmed multiple hits from a previous genome-wide pooled synthetic/lethal CRISPR dropout screen. For example, targeting genes in the cGAS/STING pathway conferred resistance to PARP7 inhibition in the NCI-H1373 responder cells, suggesting a critical dependence on this sensing pathway. In the PARP7 inhibitor-resistant HARA cells, deletion of components of innate immune-signaling (such as AIM2 and ADAR1), the NF-κB pathway, and genes involved in autophagy sensitized the cells to PARP7 inhibition. We further delineated the function of PARP7 by comparing the effects of the CRISPR perturbation across different cellular readouts such as STAT1 phosphorylation, IFN release, and proliferation. With our work, we shed light on the mechanism by which PARP7 acts as a critical suppressor of the innate immune response. Our findings demonstrate both redundancy and crosstalk between different nucleic acid-sensing pathways and may explain why some cell lines are resistant to PARP7 inhibition. Citation Format: Bin Gui, Ryan Abo, Patrick Flynn, Alvin Z. Lu, Jan-Rung Mo, Joseph M. Gozgit, Melissa M. Vasbinder, Zacharenia A. Varsamis, Andrew Santospago, Victoria M. Richon, Kevin W. Kuntz, Heike Keilhack, Timothy J. Mitchison, Mario Niepel. Investigating the mechanism of PARP7 inhibition in type I interferon signaling by arrayed CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1021.

Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Abstract 3411: High-throughput drug screening combined with a druggable target CRISPR drop-out screen identifies therapeutic vulnerabilities of BCOR-CCNB3 fusion positive sarcomas

Abstract Undifferentiated round cell sarcomas, which carry the BCOR-CCNB3 fusion gene, have traditionally been called “Ewing-like sarcomas” and have been treated with Ewing sarcoma treatment regimens. While there has been emerging evidence that these BCOR-CCNB3 sarcomas (BCS) are molecularly distinct from Ewing sarcomas, the biology of these tumors is poorly understood, and patients have few options when they do not respond to Ewing sarcoma-based therapies. Here, we report the establishment of a novel BCS cell line, which we utilized to identify a unique BCS-specific therapeutic vulnerability using a hybrid screening approach consisting of a high-throughput drug screen combined with a CRISPR/Cas9-based knock out genetic screen. The BCOR-CCNB3-positive sarcoma cell line NBC-1 was established from the tumor tissue obtained from an 18-year-old male with a soft tissue tumor of the buttock with lymph node metastasis, and the expression of the fusion BCOR-CCNB3 transcript was confirmed by RT-PCR. An in vitro high-throughput screening was performed using the L1300 library of 2570 drugs, where the cells were incubated for 48 hours with drug before cell viability was measured. This drug screen identified 148 drugs, which included 84 FDA-approved drugs. While these drugs predictably included a significant number of conventional cytotoxic chemotherapy drugs currently used to treat BCS, such as vincristine and doxorubicin, several other drug classes emerged, identifying twenty tyrosine kinase inhibitors, proteasome inhibitors such as Carfizomib, Ixazomib, and Bortezomib, and a histone deacetylase inhibitor, Panobinostat, all of which had distinctly higher cytotoxicity compared to the Ewing sarcoma cell controls. Next, we utilized a custom-made CRISPR panel consisting of 9771 sgRNAs targeting 1221 genes where we were able to identify a specific pharmacologic inhibitor in the literature. Using this library, we carried out a genetic loss-of-function screen to identify genes that are required for the proliferation of BCS. Based on these initial findings, we were able to demonstrate that BCS tumors have distinct vulnerabilities from Ewing sarcoma, suggesting that a BCS-specific therapeutic approach is necessary. Citation Format: Naoki Oike, Hiroyuki Kawashima, Akira Ogose, Takashi Ariizumi, Yudai Murayama, Hajime Umezu, Molishree Joshi, Colin Sempeck, Deandra Warker, Avery Bodlak, Naoto Endo, Masanori Hayashi. High-throughput drug screening combined with a druggable target CRISPR drop-out screen identifies therapeutic vulnerabilities of BCOR-CCNB3 fusion positive sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3411.

Integrating Enhancer Profiling and CRISPR Dropout Screen Revealed Selenoprotein Synthesis Pathway As a New Vulnerability in AML

Alterations of the epigenetic landscape and transcription are hallmarks of acute myeloid leukemia (AML) that drive leukemogenic gene expression and therefore can be exploited for therapeutic intervention. To look for such targets that harbor both an altered epigenetic feature and are genetically essential for AML cells, we performed a multi-database analysis integrating pan-cancer super enhancer landscapes with whole genome CRISPR dropout screens. Among the top targets, we discovered SEPHS2. An enhancer was present upstream of the gene marked by H3K27ac and bound by leukemogenic transcription factors including MYB, Pu.1 and RUNX1. In addition, AML cells with SEPHS2 deletion significantly dropped out in a genome wide CRISPR screen. This gene encodes a critical enzyme in the underappreciated selenoprotein synthesis pathway which was highly upregulated in TCGA AML patients compared to control blood cells from healthy individuals. Collectively, our initial bioinformatic analysis suggested that the selenoprotein synthesis pathway is a new vulnerability in AML. To test the functional requirement of the selenoprotein synthesis pathway in AML and other cells, we performed CRISPR mediated deletion of three key genes in the selenoprotein synthesis pathway, SEPHS2, SEPSECS and EEFSEC. The human AML cell lines (MOLM13, THP1 and Kasumi1), murine AML cells transformed by MLL-AF9 and human AML PDX cells all depicted a significant dependency on these genes, while proliferation of normal cord blood cells and myeloma cells (U266B1) was almost not affected. We then transplanted these cells into recipients. Deletion of SEPHS2, SEPSECS or EEFSEC significantly ameliorated AML progression, indicated by decreased AML burden and extended survival. Since selenoproteins including GPX1 and GPX4 are known antioxidants, we hypothesized that perturbing the selenoprotein synthesis pathway disrupted the redox state in AML cells. We found that deletion of SEPHS2, SEPSECS or EEFSEC elevated ROS in AML cells as demonstrated by Cell-Rox or DCF-DA staining. Western blotting revealed significantly downregulated GPX4 level and upregulated DNA damage marker γ-H2AX. Moreover, the defective proliferation was partially rescued by adding antioxidant TEMPOL. These results suggested selenoprotein synthesis pathway produced key antioxidants to balance the proper redox state and was required for AML cell proliferation. A major source of selenium is diet. Therefore, we hypothesized that consuming selenium low diet could suppress AML. We compared the survival of AML bearing mice on selenium proficient and deficient diet. The selenium deficient diet significantly extended survival, lowered GPX4 level and increased ROS in AML cells. Interestingly, normal mouse on selenium deficient diet for a 3-months period did not develop any abnormalities in CBC or bone marrow hematopoiesis. This suggests selenium deficient diet could be clinically applicable without significant side effects. Altogether, the integration of a pan-cancer enhancer landscape study with CRISPR dropout gene screen offered a powerful tool to dissect cancer targets that possessed unique enhancer features and genetic essentiality. The analysis yielded SEPHS2 and its selenoprotein synthesis pathway to be a new vulnerability in AML. The underappreciated selenoprotein synthesis pathway was key to produce antioxidant selenoproteins such as GPX1 and GPX4 to maintain a proper redox state in AML. Deleting the genes or removing selenium from diet could perturb the pathway and ameliorate the AML disease. Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

CRISPR Dropout Screens Identify DHODH, PIK3C3, and Crkl as Potential Therapeutic Targets in Acute Myeloid Leukemia

Relapsed disease is still a major challenge in AML despite recently approved targeted therapies. New therapeutic targets are needed. To identify vulnerabilities in AML, screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library (DepMap) was analyzed by examining gene knockout (KO) dependency scores in 15 AML cell lines (HEL, MV411, OCIAML2, THP1, NOMO1, EOL1, KASUMI1, NB4, OCIAML3, MOLM13, TF1, U937, F36P, AML193, P31FUJ). 438 gene KOs were identified as essential to AML cell survival in 14/15 AML cell lines. 247/438 (56%) genes had no active compound or approved drug interacting with its gene-products (DGIdb, canSAR Black) but were tractable targets based on 3D modeling or ligand-based druggability scoring (canSAR Black). This refined list of targets made up a new screening platform termed disKO. For clinical relevance, a primary AML specimen was treated with a custom-made lentiviral-based pooled CRISPR-Cas9 library including disKO targets using guide RNA's constructed against a parsed version of the druggable genome. Following transduction, PCR and oligo-sequencing were performed on Days 0, 7, and 14. Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) was used to identify gene KOs associating with AML dropout over time. Four gene KOs from disKO were identified as essential in primary AML: NUP93, DHODH, CRKL, and PIK3C3. NUP93 loss of function mutations have been associated with steroid-resistant nephrotic syndrome in patients, suggesting inhibition may be toxic. DHODH inhibitors are currently being tested in AML. PIK3C3 codes for lipid kinases involved in autophagocytosis, and PIK3C3 inhibitors are in preclinical stage of development. CRKL is a proto-oncogene encoding src homology domains (SH2, SH3), which activate RAS and JNK signaling pathways. Although CRKL is a substrate of BCR-ABL CML, its essentiality in our study was found in AML cell lines and primary AML lacking the BCR-ABL fusion. Differential gene expression analysis showed CRKL to have lower expression in AML when compared to normal hematopoietic cells. Its druggability score based on six 3D structures and 1.3 ligand z-score indicates promising pharmacologic inhibition. In conclusion, a new CRISPR-Cas9 screening method termed disKO led to the identification of three viable targets for AML: PIK3C3, DHODH, and CRKL. Current efforts are focused on target validation followed by compound screening to find inhibitors with therapeutic potential. Disclosures No relevant conflicts of interest to declare.