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This file contains supplemental methods on the PPTP scoring method.

This file contains legends to the 4 supplemental figures.

This file contains supplemental figures on in vivo xenograft responses to birinapant, a supplemental figure on mouse tolerability to birinapant, and a supplemental figure on gene expression networks. It also contains a supplemental table on patient demographics, a supplemental table on in vivo xenograft responses to birinapant, a supplemental table on individual mouse responses to birinapant, a supplemental table on in vivo birinapant efficacy in drug combinations, and a supplemental table on differentially expressed genes.

Ph-like acute lymphoblastic leukemia (ALL) is a genetically defined high-risk ALL subtype with a generally poor prognosis. In this study, we evaluated the efficacy of birinapant, a small-molecule mimetic of the apoptotic regulator SMAC, against a diverse set of ALL subtypes. Birinapant exhibited potent and selective cytotoxicity against B-cell precursor ALL (BCP-ALL) cells that were cultured ex vivo or in vivo as patient-derived tumor xenografts (PDX). Cytotoxicity was consistently most acute in Ph-like BCP-ALL. Unbiased gene expression analysis of BCP-ALL PDX specimens identified a 68-gene signature associated with birinapant sensitivity, including an enrichment for genes involved in inflammatory response, hematopoiesis, and cell death pathways. All Ph-like PDXs analyzed clustered within this 68-gene classifier. Mechanistically, birinapant sensitivity was associated with expression of TNF receptor TNFR1 and was abrogated by interfering with the TNFα/TNFR1 interaction. In combination therapy, birinapant enhanced the in vivo efficacy of an induction-type regimen of vincristine, dexamethasone, and L-asparaginase against Ph-like ALL xenografts, offering a preclinical rationale to further evaluate this SMAC mimetic for BCP-ALL treatment. Cancer Res; 76(15); 4579–91. ©2016 AACR.

PDF file - 123K, Figure S3. Effect of cell cycle on CIP2A expression.

PDF file - 66K, Supplementary Table S1. Co-relation between Chk1 and c-MYC nuclear staining in gastric cancer.

PDF file - 85K, Figure S2. Chk1 and Claspin (CLSPN) positively regulate CIP2A protein expression in human cancer cells.

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1–CIP2A–PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy. Cancer Res; 73(22); 6757–69. ©2013 AACR.

PDF file - 82K, Figure S1. Expression analysis of Chk1 and CIP2A in human malignancies.

PDF file - 90K, Supplementary Table S2. List of genes which expression significantly correlates with Chk1 and CIP2A.

PDF file - 205K

PDF file - 101K, Figure S4. In vitro and in vivo regulation of CIP2A mRNA expression by Chk1 and effect on cancer cell viability and anchorage independent growth by Chk1, Claspin and CIP2A.

Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes-associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY-box transcription factor 17 (SOX17)

The ETS transcription factor ERG is involved in several cancers including leukemias. We and others have demonstrated a direct role for ERG in leukemia initiation and maintenance of several subtypes of myeloid and lymphoid leukemias. However, ERG domains and co-factors involved in leukemogenesis remains largely uncharacterized and as a transcription factor it is undruggable. Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity. Specifically, we demonstrate that it is required for ERG-induced self-renewal and restriction of myeloid differentiation in hematopoietic progenitor cells and for initiation of leukemia in mouse transduction/transplantation models. Mechanistically, we show that P199 facilitates the interaction of ERG with the NCoR-HDAC3 co-repressor complex. Inhibition of HDAC3 reverses ERG’s restriction of myeloid differentiation and reduces human ERG-dependent leukemic burden in immunodeficient mice. Thus, the interaction of ERG with the NCoR-HDAC3 co-repressor complex is required for its oncogenic activity and modulation of this interaction may provide an opportunity for therapeutic intervention.

Human endogenous retroviruses (HERVs) form a substantial part of the human genome. However, most elements remain transcriptionally silent. Malignant transformation and epigenetic therapies may induce transcription of HERV elements. Such HERV elements could potentially trigger adaptive immune responses and provide targets for immunotherapy applications. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies. We studied 66 HERV genes reported to retain transcriptional activity and generated a library of 1,169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Through the use of DNA barcode-labeled MHC-I multimers, we identified CD8+ T cell populations recognizing 29 HERV-derived peptides, represented by 18 different HERVs. T cell recognition of HERVs was detected in half of the 34 patients analyzed and was significantly enhanced in patients compared to healthy donors. Patients with HERV-derived T cell reactivity demonstrated improved viral-antigen recognition following treatment, indicating that HERV-derived T cell recognition may contribute to the therapy-induced clinical improvement and immune reconstitution. In line with the T cell data, transcriptomic analyses revealed a significantly enhanced transcription of the HERVs in patients compared to healthy donors. DNA-demethylating therapy did not result in additional transcriptional enhancement of the HERVs evaluated for T cell recognition. Our study demonstrates substantial T cell recognition of HERVs in myeloid malignancies. These HERVs could serve as a reservoir of antigens potentially exploited as immunotherapeutic targets in malignancies with low mutational burden.