(A-C) Heatmaps of the z-score of the normalised counts of sgRNAs from the CRISPR screen over time and under B32B3 and VPR8 treatment. (A) VPR8 resistant genes. (B) VPR8 sensitivity genes. (C) Targeting regulators of H2AT120P does not modulate VPR8 response.

Multiple myeloma is a plasma cell malignancy that is susceptible to drugs targeting protein homeostasis such as thalidomide analogs and proteasome inhibitors. Thalidomide analogs modulate the activity of DDB1/CUL4 E3-ligase complexes to perturb substrate recognition and proteasomal degradation thereof. We hypothesized that the cellular pool of DDB1/CUL4-associated factors (DCAF) may mediate other essential plasma cell processes and offer new targets for therapeutic intervention. Unbiased genetic screening identified DCAF1 (also known as viral protein R-binding protein) as essential for myeloma cell survival with a multidomain structure, offering several distinct opportunities for drug development. Utilizing B32B3, a previously disclosed DCAF1 kinase inhibitor as a template, we developed a series of analogs with enhanced antimyeloma potency. As antimyeloma activity did not associate with dephosphorylation of known DCAF1 kinase substrates, we correlated drug-induced cellular phenotypes with whole-genome CRISPR/Cas9 resistance screening to further define mechanistic activity. These studies identified B32B3 analogs as microtubular destabilizing agents with potential DCAF1 kinase-independent properties and in vivo efficacy in multiple myeloma and lymphoma.

CDK4/6 inhibitors in combination with endocrine therapy are now used as front-line treatment for patients with estrogen-receptor positive (ER+) breast cancer. While this combination improves overall survival, the mechanisms of disease progression remain poorly understood. Here, we performed unbiased genome-wide CRISPR/Cas9 knockout screens using endocrine sensitive ER+ breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Our screens identified the inactivation of JNK signalling, including loss of the kinase MAP2K7, as a key driver of drug insensitivity. We developed multiple CRISPR/Cas9 knockout ER+ breast cancer cell lines (MCF-7 and T-47D) to investigate the effects of MAP2K7 and downstream MAPK8 and MAPK9 loss. MAP2K7 knockout increased metastatic burden in vivo and led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition. Furthermore, analysis of clinical datasets found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNKT183/Y185 activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies in both early-stage and metastatic ER+ breast cancer cohorts. Overall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Our data provides the pre-clinical rationale to stratify patients based on JNK pathway activity prior to receiving combination endocrine therapy and CDK4/6 inhibition.

Background Organs and tissues need to be vascularized during development. Similarly, vascularization is required to engineer thick tissues. How vessels are formed during organogenesis is not fully understood, and vascularization of engineered tissues remains a significant challenge. Methods and Results Here, we show that the extracellular matrix protein nephronectin is required for vascularization during zebrafish development as well as adult fin regeneration and is sufficient to promote mammalian vessel formation and maturation. Nephronectin a morphants and mutants exhibit diminished axial vein sprouting and posterior intersegmental vessel growth. Notably, the angiogenesis‐associated integrins itgav and itgb3.1 are coexpressed with nephronectin a in the region of the caudal vein plexus and posterior somites; nephronectin binds to integrin alpha‐V/integrin beta‐3.1 (ITGAV/ITGB3.1), and itgav morphants phenocopy nephronectin a mutants. In addition, nephronectin a mutants showed decreased vessel maturation compared with wild‐type siblings during caudal fin regeneration in adult zebrafish. Moreover, nephronectin promotes mammalian endothelial cell migration and tube formation in 2D and 3‐dimensional in vitro tissue culture. Further, nephronectin enhances vascular endothelial growth factor‐induced periaortic vascular capillary interconnectivity, vessel diameter, and vessel stability. Conclusions Collectively, our results identify nephronectin as a proangiogenic factor during embryonic development, which can be used to improve the vascularization of engineered tissues.

Purpose: Endocrine therapy in combination with CDK4/6 inhibition doubles the progression-free survival of patients with advanced ER+ breast cancer, but resistance is inevitable, leaving patients with limited treatment options. Experimental Design: We performed unbiased genome-wide CRISPR/Cas9 knockout screens using ER+ breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Screen hits were validated by CRISPR/Cas9 knockout models, mechanistic analyses and evaluation of patient samples. Results: Our screens identified the inactivation of JNK signalling, including loss of the kinase MAP2K7, as a key driver of combination resistance. We developed multiple CRISPR/Cas9 knockout ER+ breast cancer cell lines (MCF-7 and T-47D) to investigate the effects of MAP2K7, MAPK8 and MAPK9 loss. MAP2K7 knockout increased metastatic burden in vivo and led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition. Furthermore, we analysed ER+ advanced breast cancer patient cohorts and found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNKT183/Y185 activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies. Conclusions: Overall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Furthermore, our data provide a pre-clinical rationale to screen patients' tumours for JNK signalling deficiency prior to receiving combined endocrine therapy and CDK4/6 inhibition. ### Competing Interest Statement E.L provides advisory board services to AstraZeneca, Gilead, Lilly, MDS, Novartis, Pfizer, Roche, and received royalties from Walter and Eliza Hall Institute.

Adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) by ADAR1 is an essential modifier of the immunogenicity of cellular dsRNA. The role of MDA5 in sensing unedited cellular dsRNA and the downstream activation of type I interferon (IFN) signaling are well established. However, we have an incomplete understanding of pathways that modify the response to unedited dsRNA. We performed a genome-wide CRISPR screen and showed that GGNBP2, CNOT10, and CNOT11 interact and regulate sensing of unedited cellular dsRNA. We found that GGNBP2 acts between dsRNA transcription and its cytoplasmic sensing by MDA5. GGNBP2 loss prevented induction of type I IFN and autoinflammation after the loss of ADAR1 editing activity by modifying the subcellular distribution of endogenous A-to-I editing substrates and reducing cytoplasmic dsRNA load. These findings reveal previously undescribed pathways to modify diseases associated with ADAR mutations and may be determinants of response or resistance to small-molecule ADAR1 inhibitors.

RECQL4 is a member of the RecQ family of DNA helicases, essential for maintenance of genome integrity. Bi-allelic germline mutations of RECQL4 cause Rothmund-Thomson Syndrome Type 2 (RTS2), a rare inherited condition characterised by short stature, skeletal abnormalities, and elevated rates of cancers. In mice, loss or mutation of RECQL4 causes a rapid, fully penetrant bone marrow failure (BMF) phenotype. While we know RECQL4 is essential for DNA replication, how this occurs and the precise role of RECQL4 are poorly understood.To define pathways that can modify Recql4 loss of function mutations we performed a genome-wide loss of function suppressor screen in myeloid cells to identify genes, that when deleted, rescued the loss of proliferation and viability caused by RECQL4 mutation. The top candidate from the screen was Klhdc3, a substrate receptor of the Cullin2-RING E3 ubiquitin ligase (CRL2) complex. Loss of Klhdc3 restored near normal cellular viability and proliferation kinetics to cells with a RECQL4 point mutation or RECQL4 null cells, with no effect on RECQL4 wild-type cells. The re-expression of wild-type KLHDC3 in the Recql4D/D sgKlhdc3-/- myeloid cell lines caused rapid cell death. Importantly, re-expression of either a dominant-negative (DN) KLHDC3 mutant or a C-terminal deletion, both of which can still bind but not ubiquitinate their substrates, did not cause cell death, demonstrating that the rescue was specifically due to loss of KLHDC3 mediated substrate degradation.Ongoing experiments are seeking to identify the key substrates of KLHDC3 that can functionally rescue loss of RECQL4 and restore DNA replication in its absence. We are also testing if loss of KLHDC3 can prevent the BMF that characterises in vivo loss of RECQL4. These studies have identified a tractable target that can rescue and prevent phenotypes associated with the loss of RECQL4.

Abstract Objectives Autologous chimeric antigen receptor (CAR) T‐cell therapy of B‐cell malignancies achieves long‐term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR‐T manufacturing, the compromised fitness of patient‐derived T cells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR‐NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. Methods To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in‐depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR‐NK manufacturing pipeline. Results We identified adult peripheral blood‐derived NK cells to be the superior source for generating a CAR‐NK cell product because of a higher maximum yield of CAR‐expressing NK cells combined with potent natural, as well as CAR‐mediated anti‐tumor effector functions. Conclusions Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre‐ and post‐transduction and high on‐target cytotoxicity make peripheral blood‐derived NK cells a feasible and attractive CAR‐NK cell product for clinical utility.

Background Natural Killer (NK) cells can detect and eliminate virally-infected and malignant cells. As such, major efforts are underway to identify pathways that regulate these protective immune functions and exploit these for novel anti-cancer therapies. NK cells are dependent on IL-15 for their differentiation, survival and function within solid tumors, and negative regulators of this axis represent attractive drug targets. However, functional genomic screening in NK cells has been hampered by their resistance to viral transduction and efficient genome editing. Methods We developed a CRISPR-based framework for large-scale screening in primary human NK cells and derived lines as well as rapid target validation in vitro and in vivo. Human NK92 cells with Cas9 (KO) or dCas9-VP64 (activation) were transduced with a genome-wide guide library and cultured under low IL-15 or optimal growth conditions and sequenced after an extended culture period for comparison against the initial library. A druggable sub-library was also designed for screening in multiple primary human NK donors under similar limiting IL-15 conditions. Results Amongst the top enriched hits in human NK cells under limiting IL-15 availability were Cish (cytokine-induced SH2 containing protein) and oNKo-036-040. The genetic knockout of these targets identified within the multiple CRISPR screens led to enhanced NK cell proliferation assessed by EdU uptake and release of key pro-inflammatory cytokines and chemokines. Under in vitro serial killing pressure where tumor targets are replenished at regular intervals, Cish and oNKo-036-040 KO dramatically enhanced primary human NK cell cytotoxicity compared with control AAVS1 KO, which undergo functional exhaustion with repeated challenges. Furthermore, oNKo36-40 deletion alleviated NK cell immunosuppression in the presence of adenosine (NECA), prostaglandin (PGE2) and TGF-beta. oNKo-036-040 deletion further enhanced anti-BCMA CAR-NK cell function in vitro under conditions of serial tumor challenge. Target deletion in murine NK cells recapitulates human data with oNKo-037 KO enhancing IL-15 signalling and supports murine NK cell anti-metastatic function in vivo. Mechanistically, bulk RNAseq data from primary human NK cells modified with AAVS1 or oNKo-037 sgRNA indicates enhanced metabolic profile with target KO necessary for NK cell survival, persistence and effector function. Conclusions Functional genomic screens in primary human NK cells have revealed potent negative regulators of IL-15 signalling. Ablating IL-15 checkpoints in NK cells results in enhanced IL-15 signalling which translates to improved NK cell fitness and anti-tumor immunity. Cytokine checkpoints are orthogonal to traditional immune checkpoints and warrant drug discovery efforts for mono- and combination cancer immunotherapy trials. Acknowledgements We acknowledge all the staff past and present at oNKo-Innate Ethics Approval Monash University Human Ethics Approval #20458

Neuroblastoma is a deadly childhood cancer arising in the developing sympathetic nervous system. High-risk patients are currently treated with intensive chemotherapy, which is curative in only 50% of children and leaves some surviving patients with life-long side effects. microRNAs (miRNAs) are critical regulators of neural crest development and are deregulated during neuroblastoma tumorigenesis, making miRNA-based drugs an attractive therapeutic avenue. A functional screen of >1,200 miRNA mimics was conducted in neuroblastoma cell lines to discover miRNAs that sensitized cells to low doses (30% inhibitory concentration [IC30]) of doxorubicin and vincristine chemotherapy used in the treatment of the disease. Three miRNAs, miR-99b-5p, miR-380-3p, and miR-485-3p, had potent chemosensitizing activity with doxorubicin in multiple models of high-risk neuroblastoma. These miRNAs underwent genomic loss in a subset of neuroblastoma patients, and low expression predicted poor survival outcome. In vitro functional assays revealed each of these miRNAs enhanced the anti-proliferative and pro-apoptotic effects of doxorubicin. We used RNA sequencing (RNA-seq) to show that miR-99b-5p represses neuroblastoma dependency genes LIN28B and PHOX2B both in vitro and in patient-derived xenograft (PDX) tumors. Luciferase reporter assays demonstrate that PHOX2B is a direct target of miR-99b-5p. We anticipate that restoring the function of the tumor-suppressive miRNAs discovered here may be a valuable therapeutic strategy for the treatment of neuroblastoma patients.

The mechanism of action of eprenetapopt (APR-246, PRIMA-1 MET ) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis ( SLC7A11 , SHMT2 , and MTHFD1L ), as well as the enzymes required to synthesize glutathione ( GCLC and GCLM ), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.

Human papilloma viruses (HPV) are the main culprit in cervical and oropharyngeal cancers. HPV positive (+) cancers are regarded as ‘oncogene addicted’, displaying an absolute requirement for the continued expression of the oncogenes for their viability owing their survival, and thus making these genes salient targets for developing specific therapeutic agents. There is a strong association between HPV and oropharyngeal squamous cell carcinomas (OPSCC), a subset of head and neck cancers (HNCs). Alarmingly, HPV-associated OPSCC are on the rise globally, and the number of cases of HPV + OPSCCs surpasses that of cervical cancer in the USA. Here, we show that major HPV oncogenes, E6 and E7, are essential for the survival of HPV positive (+) OPSCCs, making these oncogenes salient targets for HPV-driven OPSCCs. HPV E7 is known to interact with STING, a component of the viral DNA-sensing cGAS-STING machinery which activates a pro-typical anti-viral type I interferon (IFN) response. Our recent work showed that E7 from HPV type 16 is responsible for the blockade of cGAS-STING responses in HPV + OPSCC cells. In this study, we show that CRISPR/Cas9-mediated loss of E7 from HPV + OPSCC cells, SCC2 and SCC104, restored cGAS-STING responses. Future work could involve HPV oncogene targeting leading to HPV + OPSCC tumour regression and that the combined use of STING agonists would induce favourable tumour clearance by activating appropriate anti-tumour responses.

ABSTRACTPatients with colorectal cancer (CRC) frequently develop liver metastases during the course of their disease. A substantial proportion of them receive neoadjuvant FOLFOX (5-Fluorouracil, Oxaliplatin, Leucovorin) prior to surgery in an attempt to enable successful surgical removal of their metastases and to reduce the risk of recurrence. Yet, the majority of patients progress during treatment or recur following surgery, and molecular mechanisms that contribute to FOLFOX resistance remain poorly understood. Here, using a combination of phenotypic, transcriptomic and genomic analyses of both tumor samples derived from patients with metastatic CRC and matching patient-derived tumor organoids (PDTOs), we characterize a novel FOLFOX resistance mechanism and identify inhibitors that target this mechanism to resensitize metastatic organoids to FOLFOX. Resistant PDTOs, identified afterin vitroexposure to FOLFOX, exhibited elevated expression of E2F pathway, S phase, G2/M and spindle assembly checkpoints (SAC) genes. Similar molecular features were detected in CRLM from patients with progressive disease while under neoadjuvant FOLFOX treatment, highlighting the relevance of this finding. FOLFOX resistant PDTOs displayed inactivating mutations of TP53 and exhibited transcriptional features of P53 pathway downregulation. We found that they accumulated in early S-phase and underwent significant DNA damage during FOLFOX exposure, thereafter arresting in G2/M while they repaired their DNA after FOLFOX withdrawal. In parallel, results of a large kinase inhibitor screen indicated that drugs targeting regulators of the DNA damage response, G2M checkpoint and SAC had cytotoxic effects on PDTOs generated from patients whose disease progressed during treatment with FOLFOX. Corroborating this finding, CHK1 and WEE1 inhibitors were found to synergize with FOLFOX and sensitize previously resistant PDTOs. Additionally, targeting the SAC master regulator MPS1 using empesertib after exposure to FOLFOX, when cells accumulate in G2M, was also very effective to kill FOLFOX-resistant PDTOs. Our results indicate that targeted and timely inhibition of specific cell cycle checkpoints shows great potential to improve response rates to FOLFOX in patients with metastatic CRC, for whom therapeutic alternatives remain extremely limited.