Sustained low-dose treatment with the histone deacetylase inhibitor LBH589 induces terminal differentation of osteosarcoma cells

Histone deacetylase inhibitors (HDACi) were identified nearly four decades ago based on their ability to induce cellular differentiation. However, the clinical development of these compounds as cancer therapies has focused on their capacity to induce apoptosis in hematologic and lymphoid malignancies, often in combination with conventional cytotoxic agents. In many cases, HDACi doses necessary to induce these effects result in significant toxicity. Since osteosarcoma cells express markers of terminal osteoblast differentiation in response to DNA methyltransferase inhibitors, we reasoned that the epigenetic reprogramming capacity of HDACi might be exploited for therapeutic benefit. Here, we show that continuous exposure of osteosarcoma cells to low concentrations of HDACi LBH589 (Panobinostat) over a three-week period induces terminal osteoblast differentiation and irreversible senescence without inducing cell death. Remarkably, transcriptional profiling revealed that HDACi therapy initiated gene signatures characteristic of chondrocyte and adipocyte lineages in addition to marked upregulation of mature osteoblast markers. In a mouse xenograft model, continuous low dose treatment with LBH589 induced a sustained cytostatic response accompanied by induction of mature osteoblast gene expression. These data suggest that the remarkable capacity of osteosarcoma cells to differentiate in response to HDACi therapy could be exploited for therapeutic benefit without inducing systemic toxicity

A signal-seeking Phase 2 study of olaparib and durvalumab in advanced solid cancers with homologous recombination repair gene alterations

Purpose: To determine the safety and efficacy of PARP plus PD-L1 inhibition (olaparib + durvalumab, O + D) in patients with advanced solid, predominantly rare cancers harbouring homologous recombination repair (HRR) defects. Patients and methods: In total, 48 patients were treated with O + D, 16 with BRCA1/2 alterations (group 1) and 32 with other select HRR alterations (group 2). Overall, 32 (66%) patients had rare or less common cancers. The primary objective of this single-arm Phase II trial was a progression-free survival rate at 6 months (PFS6). Post hoc exploratory analyses were conducted on archival tumour tissue and serial bloods. Results: The PFS6 rate was 35% and 38% with durable objective tumour responses (OTR) in 3(19%) and 3(9%) in groups 1 and 2, respectively. Rare cancers achieving an OTR included cholangiocarcinoma, perivascular epithelioid cell (PEComa), neuroendocrine, gallbladder and endometrial cancer. O + D was safe, with five serious adverse events related to the study drug(s) in 3 (6%) patients. A higher proportion of CD38 high B cells in the blood and higher CD40 expression in tumour was prognostic of survival. Conclusions: O + D demonstrated no new toxicity concerns and yielded a clinically meaningful PFS6 rate and durable OTRs across several cancers with HRR defects, including rare cancers

Amplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers

Translational biology of osteosarcoma

For the past 30 years, improvements in the survival of patients with osteosarcoma have been mostly incremental. Despite evidence of genomic instability and a high frequency of chromothripsis and kataegis, osteosarcomas carry few recurrent targetable mutations, and trials of targeted agents have been generally disappointing. Bone has a highly specialized immune environment and many immune signalling pathways are important in bone homeostasis. The success of the innate immune stimulant mifamurtide in the adjuvant treatment of non-metastatic osteosarcoma suggests that newer immune-based treatments, such as immune checkpoint inhibitors, may substantially improve disease outcome

Studying the role of the immune system on the antitumor activity of a Hedgehog inhibitor against murine osteosarcoma

Recent evidence demonstrates that the efficacy of conventional anticancer therapies including chemotherapy requires a functional immune system. Here, we addressed the possibility that the antitumor effect of a selective Smoothened antagonist and Hedgehog (Hh) pathway inhibitor (LDE225), a promising anticancer drug, might at least partially depend on the immune system. To this aim, we used tumor cell lines derived from a murine model of radiation-induced osteosarcoma. In vitro treatment of osteosarcoma cells with LDE225 resulted in a decreased ability of tumor cells to proliferate, but had no effect on their viability. Flow cytometry analysis demonstrated that LDE225-treatment did not detectably modulate the immunogenicity of tumor cells. Moreover, LDE225 did not display any pro-apoptotic properties on osteosarcoma cells, highlighting that its antitumor profile mainly derives from a cytostatic effect. Furthermore, calreticulin exposure, a key feature of immunogenic cell death, was not provoked by LDE225, neither alone nor combined with recognized immunogenic drugs. Finally, the oral administration of LDE225 to osteosarcoma-bearing mice did significantly delay the tumor growth even in an immunocompromised setting. These data suggest that inhibiting Hh signaling can control osteosarcoma cell proliferation but does not modulate the immunogenic profile of these cells

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing

Abstract Control of CRISPR/Cas12a trans-cleavage is crucial for biosensor development. Here, we show that small circular DNA nanostructures which partially match guide RNA sequences only minimally activate Cas12a ribonucleoproteins. However, linearizing these structures restores activation. Building on this finding, an Autocatalytic Cas12a Circular DNA Amplification Reaction (AutoCAR) system is established which allows a single nucleic acid target to activate multiple ribonucleoproteins, and greatly increases the achievable reporter cleavage rates per target. A rate-equation-based model explains the observed near-exponential rate trends. Autocatalysis is also sustained with DNA nanostructures modified with fluorophore-quencher pairs achieving 1 aM level (<1 copy/μL) DNA detection (106 times improvement), without additional amplification, within 15 min, at room temperature. The detection range is tuneable, spanning 3 to 11 orders of magnitude. We demonstrate 1 aM level detection of SNP mutations in circulating tumor DNA from blood plasma, genomic DNA (H. Pylori) and RNA (SARS-CoV-2) without reverse transcription as well as colorimetric lateral flow tests of cancer mutations with ~100 aM sensitivity

Infiltrating myeloid cells drive osteosarcoma progression via GRM4 regulation of IL23

The glutamate metabotropic receptor 4 (GRM4) locus is linked to susceptibility to human osteosarcoma, through unknown mechanisms. We show that Grm4 gene– targeted mice demonstrate accelerated radiation-induced tumor development to an extent comparable with Rb1 mice. GRM4 is expressed in myeloid cells, selectively regulating expression of IL23 and the related cytokine IL12. Osteosarcoma-conditioned media induce myeloid cell Il23 expression in a GRM4-dependent fashion, while suppressing the related cytokine Il12. Both human and mouse osteosarcomas express an increased IL23:IL12 ratio, whereas higher IL23 expression is associated with worse survival in humans. Consistent with an oncogenic role, Il23 mice are strikingly resistant to osteosarcoma development. Agonists −/− of GRM4 or a neutralizing antibody to IL23 suppressed osteosarcoma growth in mice. These findings identify a novel, druggable myeloid suppressor pathway linking GRM4 to the proinflammatory IL23/IL12 axis. SIGNIFICANCE: Few novel systemic therapies targeting osteosarcoma have emerged in the last four decades. Using insights gained from a genome-wide association study and mouse modeling, we show that GRM4 plays a role in driving osteosarcoma via a non–cell-autonomous mechanism regulating IL23, opening new avenues for therapeutic intervention

Molecular Profiling of Giant Cell Tumor of Bone and the Osteoclastic Localization of Ligand for Receptor Activator of Nuclear Factor κB

Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor κB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease