Integrated RNAi screening identifies the NEDDylation pathway as a synergistic partner of azacytidine in acute myeloid leukemia

Treatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis

As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

P04.04 Optimizing dasatinib for glioblastoma treatment

BACKGROUND Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis, even with surgical and chemoradiotherapy. Expression profiling studies classify IDH-wildtype Glioblastoma into three subtypes: Proneural (PN), mesenchymal (MES) and classical (CL). A promising target to inhibit in Glioblastoma is the non-receptor tyrosine kinase and proto-oncogene SRC. After robust pre-clinical results, SRC inhibitors like dasatinib did not improve survival of Glioblastoma patients after recurrence in clinical trials. MATERIAL AND METHODS Consolidating efforts to personalize cancer therapy, we use in silico analyses backed by in vitro and in vivo experiments on Glioblastoma stem-like cells (GSCs) derived from primary patient tumors to present a novel stratification strategy for dasatinib therapy in glioblastoma. To further tackle dasatinib resistance in GSCs, a pooled shRNA library against 5000 genes was combined with dasatinib to identify genes whose knockdown sensitizes GSCs to dasatinib. This was integrated with proteomics and phosphoproteomics data of dasatinib inhibited GSCs. RESULTS We found MES tumors with high expression of SERPINH1 to be sensitive to dasatinib inhibition, compared to the CL and PN subtypes. Interestingly, SRC phosphorylation status did not predict the efficacy of dasatinib inhibition. Computational analyses integrating data from the loss-of-function dropout viability screen and proteomics/phosphoproteomics using a novel modification of the SamNet algorithm identified Wee1, a tyrosine kinase involved in cell-cycle signaling, as a potential combination inhibition target with dasatinib. Further validation experiments showed a robust synergistic effect through combination of dasatinib and the wee1 inhibitor, MK-1775 in PN GSCs. CONCLUSION This study highlights strategies to optimize dasatinib treatment in different glioblastoma subtypes. While the stratification of patients harboring mesenchymal glioblastoma with SERPINH1 overexpression could provide an option in this particular subtype, combining dasatinib or other SRC inhibitors with Wee1 inhibitors could present an additional possibility for treating resistant proneural tumors

Combined genetic and chemical screens indicate protective potential for EGFR inhibition to cardiomyocytes under hypoxia

The return of blood flow to ischemic heart after myocardial infarction causes ischemia–reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia–reperfusion injury. Here we screened for targets for the treatment of ischemia–reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein–protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction.

Integrated RNAi Screening Identifies The NEDDylation Pathway As A Synergistic Partner of Azacytidine In Acute Myeloid Leukemia

Treatment of Acute Myeloid Leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Azacytidine and NEDD8 inhibitors are currently undergoing clinical trials in combination with azacytidine. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12

The tumour suppressor FBW7 is a substrate adaptor for the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), that targets several oncoproteins for proteasomal degradation. FBW7 is widely mutated and FBW7 protein levels are commonly downregulated in cancer. Here, using an shRNA library screen, we identify the HECT-domain E3 ubiquitin ligase TRIP12 as a negative regulator of FBW7 stability. We find that SCFFBW7-mediated ubiquitylation of FBW7 occurs preferentially on K404 and K412, but is not sufficient for its proteasomal degradation, and in addition requires TRIP12-mediated branched K11-linked ubiquitylation. TRIP12 inactivation causes FBW7 protein accumulation and increased proteasomal degradation of the SCFFBW7 substrate Myeloid Leukemia 1 (MCL1), and sensitizes cancer cells to anti-tubulin chemotherapy. Concomitant FBW7 inactivation rescues the effects of TRIP12 deficiency, confirming FBW7 as an essential mediator of TRIP12 function. This work reveals an unexpected complexity of FBW7 ubiquitylation, and highlights branched ubiquitylation as an important signalling mechanism regulating protein stability.

Non-targeting control for MISSION® shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

In parallel with the expansion of RNA interference techniques evidence has been accumulating that RNAi analyses may be seriously biased due to off-target effects of gene-specific shRNAs. Our work points to another possible source of misinterpretations of shRNA-based data – off-target effects of non-targeting shRNA. We found that one such control for the MISSION® library (commercialized TRC library), SHC016, is cytotoxic. Using a lentiviral vector with inducible expression of SHC016 we proved that this shRNA induces apoptosis in murine cells and, depending on p53 status, senescence or mitotic catastrophe in human tumor cell lines. We identified SNRPD3, a core spliceosomal protein, as a major SHC016 target in several cell lines and confirmed in A549 and U251 cell lines that CRISPRi-knockdown of SNRPD3 mimics the effects of SHC016 expression. Our finding disqualifies non-targeting SHC016 shRNA and adds a new premise to the discussion about the sources of uncertainty of RNAi results.

KIAA1841, a novel SANT and BTB domain-containing protein, inhibits class switch recombination

Class switch recombination (CSR) enables B cells to produce different immunoglobulin isotypes and mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using a shRNA library screen in a mouse B cell line, we have identified the novel protein KIAA1841 (NM_027860) as a negative regulator of CSR. KIAA1841 is an uncharacterized protein of 82kD containing SANT and BTB domains. The BTB domain of KIAA1841 exhibited characteristic properties such as self-dimerization and interaction with co-repressor proteins. Overexpression of KIAA1841 inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR.

Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.