Pharmacological disruption of the Notch transcription factor complex.

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia.

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2-inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.

Pediatric T-ALL type-1 and type-2 relapses develop along distinct pathways of clonal evolution.

The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia.

Human Colon-on-a-Chip Enables Continuous In Vitro Analysis of Colon Mucus Layer Accumulation and Physiology.

BACKGROUND & AIMS: The mucus layer in the human colon protects against commensal bacteria and pathogens, and defects in its unique bilayered structure contribute to intestinal disorders, such as ulcerative colitis. However, our understanding of colon physiology is limited by the lack of in vitro models that replicate human colonic mucus layer structure and function. Here, we investigated if combining organ-on-a-chip and organoid technologies can be leveraged to develop a human-relevant in vitro model of colon mucus physiology. METHODS: A human colon-on-a-chip (Colon Chip) microfluidic device lined by primary patient-derived colonic epithelial cells was used to recapitulate mucus bilayer formation, and to visualize mucus accumulation in living cultures noninvasively. RESULTS: The Colon Chip supports spontaneous goblet cell differentiation and accumulation of a mucus bilayer with impenetrable and penetrable layers, and a thickness similar to that observed in the human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip showed that stimulation of the colonic epithelium with prostaglandin E2, which is increased during inflammation, causes rapid mucus volume expansion via an Na-K-Cl cotransporter 1 ion channel-dependent increase in its hydration state, but no increase in de novo mucus secretion. CONCLUSIONS: This study shows the production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real time noninvasively. The Colon Chip may offer a new preclinical tool to analyze the role of mucus in human intestinal homeostasis as well as diseases, such as ulcerative colitis and cancer.

Chromatin accessibility landscape of pediatric T-lymphoblastic leukemia and human T-cell precursors.

We aimed at identifying the developmental stage at which leukemic cells of pediatric T-ALLs are arrested and at defining leukemogenic mechanisms based on ATAC-Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T-cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surrounding SAE1 was identified to best distinguish thymic developmental stages suggesting a potential role of SUMOylation in T-cell development. Deconvolution using signature regions revealed that T-ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed by TF-binding motif profiles. We integrated ATAC-Seq and RNA-Seq and found DAB1, a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper-accessible in T-ALLs. DAB1-negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper-accessible binding sites for SPI1 (PU.1), a TF crucial for normal T-cell maturation. In conclusion, our analyses of chromatin accessibility and TF-binding motifs showed that pediatric T-ALL cells are most similar to immature thymic precursors, indicating an early developmental arrest.

Author Correction: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

PDX models recapitulate the genetic and epigenetic landscape of pediatric T-cell leukemia.

We compared 24 primary pediatric T-cell acute lymphoblastic leukemias (T-ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient-derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome-wide chromatin accessibility (ATAC-Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T-ALL-derived PDX models.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Cell and molecular determinants of in vivo efficacy of the BH3 mimetic ABT-263 against pediatric acute lymphoblastic leukemia xenografts.

PURPOSE: Predictive biomarkers are required to identify patients who may benefit from the use of BH3 mimetics such as ABT-263. This study investigated the efficacy of ABT-263 against a panel of patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts and utilized cell and molecular approaches to identify biomarkers that predict in vivo ABT-263 sensitivity. EXPERIMENTAL DESIGN: The in vivo efficacy of ABT-263 was tested against a panel of 31 patient-derived ALL xenografts composed of MLL-, BCP-, and T-ALL subtypes. Basal gene expression profiles of ALL xenografts were analyzed and confirmed by quantitative RT-PCR, protein expression and BH3 profiling. An in vitro coculture assay with immortalized human mesenchymal cells was utilized to build a predictive model of in vivo ABT-263 sensitivity. RESULTS: ABT-263 demonstrated impressive activity against pediatric ALL xenografts, with 19 of 31 achieving objective responses. Among BCL2 family members, in vivo ABT-263 sensitivity correlated best with low MCL1 mRNA expression levels. BH3 profiling revealed that resistance to ABT-263 correlated with mitochondrial priming by NOXA peptide, suggesting a functional role for MCL1 protein. Using an in vitro coculture assay, a predictive model of in vivo ABT-263 sensitivity was built. Testing this model against 11 xenografts predicted in vivo ABT-263 responses with high sensitivity (50%) and specificity (100%). CONCLUSION: These results highlight the in vivo efficacy of ABT-263 against a broad range of pediatric ALL subtypes and shows that a combination of in vitro functional assays can be used to predict its in vivo efficacy.