We have developed and validated a highly specific, versatile antibody to the extracellular domain of human LGR5 (α-LGR5). α-LGR5 detects LGR5 overexpression in >90% of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B-ALL tumour cells and was used to generate an Antibody-Drug Conjugate (α-LGR5-ADC), Bispecific T-cell Engager (α-LGR5-BiTE) and Chimeric Antigen Receptor (α-LGR5-CAR). α-LGR5-ADC was the most effective modality for targeting LGR5+ cancer cells in vitro and demonstrated potent anti-tumour efficacy in a murine model of human NALM6 pre-B-ALL driving tumour attrition to less than 1% of control treatment. α-LGR5-BiTE treatment was less effective in the pre-B-ALL cancer model yet promoted a twofold reduction in tumour burden. α-LGR5-CAR-T cells also showed specific and potent LGR5+ cancer cell killing in vitro and effective tumour targeting with a fourfold decrease in pre-B-ALL tumour burden relative to controls. Taken together, we show that α-LGR5 can not only be used as a research tool and a biomarker but also provides a versatile building block for a highly effective immune therapeutic portfolio targeting a range of LGR5-expressing cancer cells.

Loss-of-function mutations in the gene encoding the acetyltransferase CREBBP have been reported in numerous cancers but are particularly frequent in lymphoid malignancies. However, the functional significance of CREBBP loss in transformation and disease progression, most likely through cooperation with secondary genetic hits, has not yet been fully unravelled. Similarly, the contribution of the initial cell population sustaining CREBBP loss in the course of disease remains elusive. Here, we developed a new lymphoma mouse model integrating Crebbp loss at various stages of B cell development with a transposon-based insertional mutagenesis system. We demonstrated that Crebbp loss from the HSPC compartment resulted in an aggressive DLBCL-like disease, recapitulating well-characterised histological and molecular features of the human disease, as well as the recently described enhanced CD24 expression. More importantly, we identified candidate genes functionally equivalent to patient mutated genes. Those genes, mainly related to B cell development and cellular signalling, may represent novel therapeutic targets. Overall, this new model provides a powerful resource in which to conduct future mechanistic and therapeutic studies.### Competing Interest StatementThe authors have declared no competing interest.

B-cell acute lymphoblastic leukemia (B-ALL) is a leading cause of death in childhood and outcomes in adults remain dismal. There is therefore an urgent clinical need for therapies that target the highest risk cases. Mutations in the histone acetyltransferase CREBBP associate with high-risk features in B-ALL and have been implicated in chemoresistance. We performed a targeted drug screen in isogenic human cell lines, identifying a number of actionable small molecules that specifically target CREBBP -mutated B-ALL. The most potent was the BCL2 inhibitor Venetoclax, which acts through a non-canonical mechanism resulting in ferroptotic cell death. CREBBP -mutated cell lines showed differences in cell-cycle, metabolism and response to oxidative stress. Lastly, we demonstrate that small-molecule inhibition of CREBBP sensitizes B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in-vitro and in-vivo , providing a potential novel drug combination for broader clinical translation in B-ALL. ### Competing Interest Statement NN is a former employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. NN received payments from WEHI related to venetoclax.

Supplemental Movie 4: Animated EVAC streamlines (grey lines), showing the path taken by interstitial fluid, overlaid on vascular perfusion measurements (colour scale, measured in vivo using arterial spin labelling) and the location of blood vessels (yellow structures, measured ex vivo using micro-CT). The data were acquired in an SW1222 colorectal carcinoma tumour xenograft.

Abstract Several distinct fluid flow phenomena occur in solid tumors, including intravascular blood flow and interstitial convection. Interstitial fluid pressure is often raised in solid tumors, which can limit drug delivery. To probe low-velocity flow in tumors resulting from raised interstitial fluid pressure, we developed a novel MRI technique named convection-MRI, which uses a phase-contrast acquisition with a dual-inversion vascular nulling preparation to separate intra- and extravascular flow. Here, we report the results of experiments in flow phantoms, numerical simulations, and tumor xenograft models to investigate the technical feasibility of convection-MRI. We observed a significant correlation between estimates of effective fluid pressure from convection-MRI with gold-standard, invasive measurements of interstitial fluid pressure in mouse models of human colorectal carcinoma. Our results show how convection-MRI can provide insights into the growth and responsiveness to vascular-targeting therapy in colorectal cancers. Significance: A noninvasive method for measuring low-velocity fluid flow caused by raised fluid pressure can be used to assess changes caused by therapy. Cancer Res; 78(7); 1859–72. ©2018 AACR.

Supplemental Movie 3: Animated EVAC streamlines (grey lines), showing the path taken by interstitial fluid, overlaid on vascular perfusion measurements (colour scale, measured in vivo using arterial spin labelling) and the location of blood vessels (yellow structures, measured ex vivo using micro-CT). The data were acquired in an LS174T colorectal carcinoma tumour xenograft.

Supplementary Table 1: List of the main targets and number of probes included in the small-molecule inhibitors library (related to Fig. 1)

Supplementary information provided in this work.

Supplementary Table 3: Mutations identified by exome sequencing in Liu et al., 2017 T-ALL cohort (related to Fig. 5G)

Supplemental Movie 5: Interstitial convection, measured using EVAC-MRI and animated using a particle simulation (left), compared with MRI signal enhancement with time, following injection with a contrast agent (Gd-DTPA), in an LS174T tumour.

Supplemental Movie 1: Schematic movie illustrating the physical principles underpinning the convection-MRI sequence

<p>Supplemental Movie 2: Animated EVAC streamlines (grey lines), showing the path taken by interstitial fluid, overlaid on vascular perfusion measurements (colour scale, measured in vivo using arterial spin labelling) and the location of blood vessels (yellow structures, measured ex vivo using micro-CT). The data were acquired in an LS174T colorectal carcinoma tumour xenograft.</p>

Additional data describing the reproducibility of convectionMRI data, and a figure showing the convectionMRI acquisition sequence.

Supplementary Table 2: Cell viability data from the small-molecule drug screening in EZH2-WT and EZH2-KO1 cells (related to Fig. 1)

High-resolution copy of Figure 6b. Fluid flow measured in vivo with convectionMRI is shown as grey streamlines and perfusion is shown as a colorscale. The location of blood vessels is represented by yellow volume renderings, acquired using ex vivo microvascular casting and imaged with micro-CT.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a malignant disorder of immature B lineage immune progenitors and is the commonest cancer in children. Despite treatment advances it remains a leading cause of death in childhood and response rates in adults remain poor. A preleukemic state predisposing children to BCP-ALL frequently arises in utero , with an incidence far higher than that of transformed leukemia, offering the potential for early intervention to prevent disease. Understanding the natural history of this disease requires an appreciation of how cell-extrinsic pressures, including microenvironment, immune surveillance and chemotherapy direct cell-intrinsic genetic and epigenetic evolution. In this review, we outline how microenvironmental factors interact with BCP-ALL at different stages of tumorigenesis and highlight emerging therapeutic avenues.

Leucine-rich repeat-containing G-protein receptor 5 (LGR5) has been characterised as a stem cell and cancer stem cell marker. Previous analyses of LGR5 transcript levels indicate high level expression discriminates malignancies such as colorectal cancer (CRC) and pre-B acute lymphoblastic leukaemia (pre-B ALL) from healthy tissues suggesting LGR5 protein expression may provide a molecular handle for prognosis and treatment. We have developed highly specific, high affinity antibodies to the extracellular domain of human LGR5 (α-LGR5) that detect high LGR5 protein levels in colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL. In contrast, there is low to undetectable levels of LGR5 protein in normal colon and rectal epithelia, liver, ovarian tissues, brain and immune cell types. LGR5 is rapidly internalised from the plasma membrane and trafficked to intracellular vesicular compartments including lysosomes. Treatment of high LGR5-expressing CRC and pre-B ALL cancer cell lines with an antibody-drug conjugate version of α-LGR5 (α-LGR5-ADC) lead to effective cell killing at nanomolar concentrations. Interventional treatment of pre-B ALL tumours with α-LGR5-ADC in vivo led to rapid tumour attrition. We further demonstrated the therapeutic utility of humanised α-LGR5 by using the corresponding scFv fragment for the generation of α-LGR5 chimeric antigen receptors (CARs) and a Bispecific T cell Engager (BiTE). α-LGR5-CAR-NK cells were effective at killing LGR5-expressing cells while α-LGR5/α-CD3 BiTEs induce T cell activation and killing of NALM6 cells by cytotoxic CD8 + T cells. Taken together, this study establishes α-LGR5-based therapeutic modalities that effectively discriminate and target CRC, HCC and pre-B ALL tumour cells. One Sentence Summary We generated novel antibodies against the cancer cell marker LGR5, validated diagnostic use in prioritizing specific cancer types for targeting, and developed antibody-based therapeutics.

De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8(+/-) NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells - two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cellcycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8(+/-) developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.

Several studies have documented aberrant RNA editing patterns across multiple tumors across large patient cohorts from The Cancer Genome Atlas (TCGA). However, studies on understanding the role of RNA editing in acute myeloid leukemia (AML) have been limited to smaller sample sizes. Using high throughput transcriptomic data from the TCGA, we demonstrated higher levels of editing as a predictor of poor outcome within the AML patient samples. Moreover, differential editing patterns were observed across individual AML genotypes. We also could demonstrate a negative association between the degree of editing and mRNA abundance for some transcripts, identifying the potential regulatory potential of RNA-editing in altering gene expression in AML. Further edQTL analysis suggests potential cis-regulatory mechanisms in RNA editing variation. Our work suggests a functional and regulatory role of RNA editing in the pathogenesis of AML and we extended our analysis to gain insight into the factors influencing altered levels of editing.

The ETV6-RUNX1 onco-fusion arises in utero , initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.