SUMMARY Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that, in order to be realized, must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276-ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276-ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276 targeted dual-compartment ablation could aid in development of highly selective broad-acting anti-cancer therapies.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2cko/ko mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2ko/ko cells. PARP1 deficiency does not restore HR in Brca2ko/ko cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2cko/cko mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.

Toll-like receptor 4 (TLR4) plays a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a TLR4-induced gene. Here, we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3β. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide (LPS). FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of TLR4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling.

No abstract

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the world. Pancreatic cancer can be localized, locally advanced or metastatic. The median 1- and 5-year survival rates are 25% and 6%, respectively. Epigenetic modifications such as DNA methylation play a significant role during both normal human development and cancer progression. To investigate epigenetic regulation of genes in the tumor-initiating population of pancreatic cancer cells, which are also termed cancer stem cells (CSCs), we conducted epigenetic arrays in PANC1 and HPAC pancreatic cancer cell lines and compared the global DNA methylation status of CpG promoters in invasive cells, demonstrated to be CSCs, to their non-invasive counterparts, or non-CSCs. Our results suggested that the NF-κB pathway is one of the most activated pathways in pancreatic CSCs. In agreement with this, we determined that upon treatment with NF-κB pathway inhibitors, the stem cell-like properties of cells are significantly disrupted. Moreover, SOX9, demethylated in CSCs, is shown to play a crucial role in the invasion process. Additionally, we found a potential NF-κB binding site located in the SOX9 promoter, and determined that the NF-κB subunit p65 positively regulates SOX9 expression by binding to its promoter directly. This interaction can be efficiently blocked by NF-κB inhibitors. Thus, our work establishes a link between the classical NF-κB signaling transduction pathway and the invasiveness of pancreatic CSCs, which may result in the identification of novel signals and molecules that function at an epigenetic level, and could potentially be targeted for pharmaceutical investigations and clinical trials.

Inhibitors of DNA binding (Id) family members are key regulators of cellular differentiation and proliferation. These activities are related to the ability of Id proteins to antagonize E proteins and other transcription factors. IntroductionHematopoiesis, the process by which mature blood cells of distinct lineages are produced from multipotent hematopoietic stem cells (HSCs), is a highly orchestrated process, involving a hierarchy of progenitors with progressively restricted developmental potential. 1,2 Transcription factors play a key role in hematopoietic lineage commitment, depending on their expression levels as well as their interactions. [3][4][5] For example, the importance of E proteins in the regulation of lymphocyte development was firmly established in the studies of 2 independently produced E2A knock-out mouse strains, which display a block in B-cell development and perturbed T-cell development. 6-8 During myeloid development, PU.1 and C/EBP␣ are up-regulated in the granulocyte/macrophage progenitors (GMPs) during granulocyte and macrophage development, and down-regulated in the megakaryocyte/erythrocyte progenitors (MEPs). 4,9 They cooperate in the regulation of a number of myeloid-specific genes, such as the granulocyte/macrophage colonystimulating factor receptor ␣ (GM-CSFR␣), macrophage CSF receptor (M-CSFR), and granulocyte CSF receptor (G-CSFR). [10][11][12][13] In comparison, GATA-1 and its cofactor FOG-1, which are required for erythroid differentiation, are up-regulated in MEPs and down-regulated in GMPs. 4,14,15 In addition, the antagonistic interaction between PU.1 and GATA-1 is critical in initiating the myeloid versus the erythroid program. 16,17 Id2 is a member of the inhibitor of DNA binding protein (Id) family. Id proteins play important roles in regulating cell proliferation, differentiation, and apoptosis. [18][19][20] Mechanistically, Id proteins act as dominant-negative regulators of other transcription factors and render them unable to bind DNA and regulate transcription. Id proteins bind to ubiquitously expressed bHLH transcription factors E proteins, and prevent E protein homodimerization or heterodimerization with tissue-restricted basic HLH (bHLH) proteins. 21,22 In addition to their interaction with E proteins, Id proteins have also been shown to interact with other transcription factors, including transcription factors from the ETS family, Pax family, and retinoblastoma protein RB. [23][24][25] As negative regulators of E proteins, Id proteins have been implicated in the lymphocyte proliferation and developmental progression. 26,27 Overexpression of Id1, Id2, or Id3 has similar effects on lymphocyte development. [28][29][30][31] However, which Id protein plays a physiologic role during lymphocyte development is not clear. In this study, by analyzing Id2 knock-out mice and retroviral transduced hematopoietic progenitors, we demonstrated that Id2 is an intrinsic negative regulator of B-cell development. Furthermore, we identified novel Id2 function in erythroid development....

Loss of Sirt1 causes increased Hoxa9 expression and expansion of HSPC subsets under hematopoietic stress, resulting in increased DNA damage and exhaustion of long-term progenitors.