The challenging riddle about the janus‐type role of hsp60 and related extracellular vesicles and miRNAs in carcinogenesis and the promises of its solution

Hsp60 is one of the most ancient and evolutionarily conserved members of the chaperoning system. It typically resides within mitochondria, in which it contributes to maintaining the organelle’s proteome integrity and homeostasis. In the last few years, it has been shown that Hsp60 also occurs in other locations, intracellularly and extracellularly, including cytosol, plasmacell membrane, and extracellular vesicles (EVs). Consequently, non‐canonical functions and interacting partners of Hsp60 have been identified and it has been realized that it is a hub molecule in diverse networks and pathways and that it is implicated, directly or indirectly, in the development of various pathological conditions, the Hsp60 chaperonopathies. In this review, we will focus on the multi‐faceted role of this chaperonin in human cancers, showing the contribution of intra‐ and extracellular Hsp60 in cancer development and progression, as well as the impact of miRNA‐mediated regulation of Hsp60 in carcinogenesis. There are still various aspects of this intricate biological scenario that are poorly understood but ongoing research is steadily providing new insights and we will direct attention to them. For instance, we will highlight the possible applications of the Hsp60 involvement in carcinogenesis not only in diagnosis, but also in the development of specific anti‐cancer therapies centered on the use of the chaperonin as therapeutic target or agent and depending on its role, pro‐ or anti‐tumor

A multifunctional platform for the production and customization of polymer-based microneedle devices

Polymer microneedles (MNs) have significant potential for use in transdermal delivery and diagnostics applications due to their low cost, versatility, and compatibility with medical grade materials and industrial manufacturing processes. These polymers can also have a wide range of different and desirable properties such as biocompatibility, degradability, and flexibility. To facilitate rapid development of these devices, a multifunctional manufacturing process, easily adaptable to a range of different materials and use cases, would be highly beneficial for research and prototyping purposes. With that in mind, we have developed a multifunctional platform that may be used to produce sharp-tipped microneedle arrays with a variety of substrate materials, mechanical characteristics, electrical properties, and diagnostic functionalities. The paper first presents an outline of the platform concept and the double-sided moulding process that lies at its core, followed by a description of the various add-on steps that are used to customise the geometrical, mechanical, electrical, and functional aspects of the arrays. Finally, we illustrate the versatility of the platform with three exemplars, namely a solid, electrochemically active MN sensor for biomarker diagnostics, a fabric-backed, flexible MN electrode for biopotential monitoring, and a biodegradable array for transdermal drug delivery

Entospletinib with decitabine in acute myeloid leukemia with mutant TP53 or complex karyotype: A phase 2 substudy of the Beat AML Master Trial

BackgroundPatients with acute myeloid leukemia (AML) who have tumor protein p53 (TP53) mutations or a complex karyotype have a poor prognosis, and hypomethylating agents are often used. The authors evaluated the efficacy of entospletinib, an oral inhibitor of spleen tyrosine kinase, combined with decitabine in this patient population.MethodsThis was a multicenter, open-label, phase 2 substudy of the Beat AML Master Trial (ClinicalTrials.gov identifier NCT03013998) using a Simon two-stage design. Eligible patients aged 60 years or older who had newly diagnosed AML with mutations in TP53 with or without a complex karyotype (cohort A; n = 45) or had a complex karyotype without TP53 mutation (cohort B; n = 13) received entospletinib 400 mg twice daily with decitabine 20 mg/m2 on days 1-10 every 28 days for up to three induction cycles, followed by up to 11 consolidation cycles, in which decitabine was reduced to days 1-5. Entospletinib maintenance was given for up to 2 years. The primary end point was complete remission (CR) and CR with hematologic improvement by up to six cycles of therapy.ResultsThe composite CR rates for cohorts A and B were 13.3% (95% confidence interval, 5.1%-26.8%) and 30.8% (95% confidence interval, 9.1%-61.4%), respectively. The median duration of response was 7.6 and 8.2 months, respectively, and the median overall survival was 6.5 and 11.5 months, respectively. The study was stopped because the futility boundary was crossed in both cohorts.ConclusionsThe combination of entospletinib and decitabine demonstrated activity and was acceptably tolerated in this patient population; however, the CR rates were low, and overall survival was short. Novel treatment strategies for older patients with TP53 mutations and complex karyotype remain an urgent need

Aequorin-based measurements of intracellular Ca(2+)-signatures in plant cells

Due to the involvement of calcium as a main second messenger in the plant signaling pathway, increasing interest has been focused on the calcium signatures supposed to be involved in the patterning of the specific response associated to a given stimulus. In order to follow these signatures we described here the practical approach to use the non-invasive method based on the aequorin technology. Besides reviewing the advantages and disadvantages of this method we report on results showing the usefulness of aequorin to study the calcium response to biotic (elicitors) and abiotic stimuli (osmotic shocks) in various compartments of plant cells such as cytosol and nucleus

A Study to Assess the Efficacy of Enasidenib and Risk-Adapted Addition of Azacitidine in Newly Diagnosed IDH2-Mutant AML

Enasidenib (ENA) is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML substudy, we applied a risk-adapted approach to assess the efficacy of ENA monotherapy for patients aged ≥60 years with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated that mutant IDH2 was in the dominant leukemic clone. Patients for whom ENA monotherapy did not induce a complete remission (CR) or CR with incomplete blood count recovery (CRi) enrolled in a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to ENA alone demonstrated efficacy, with a composite complete response (cCR) rate (CR/CRi) of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and 1 dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of ENA monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML. This trial is registered at www.clinicaltrials.gov as #NCT03013998

Stimulation of macrophage urokinase expression by polyanions is protein kinase C-dependent and requires protein and RNA synthesis

Highly charged polyanionic ligands of the scavenger receptor trigger macrophage secretion of urokinase-type plasminogen activator (uPA). In experiments reported here, we have investigated the intracellular and extracellular regulation of polyanion-induced macrophage plasminogen activation. Exposure of a macrophage cell line (RAW264.7) to either fucoidan or phorbol myristate acetate (PMA) stimulates the secretion of uPA, whereas calcium ionophore or dibutyryl cyclic AMP had no effect. Moreover, preincubation of macrophages with inhibitors of protein kinase C reduced (50-60%) the ability of both fucoidan and PMA to trigger the secretion of uPA, whereas aspirin and eicosatetraenoic acid had no effect. Both PMA and fucoidan treatment of RAW264.7 cells resulted in a rapid and transient increase in the steady state levels of uPA mRNA. However, in marked contrast to that observed with PMA, fucoidan-induced expression of RAW264.7 uPA activity was partially insensitive to cycloheximide and actinomycin D. In addition, fucoidan-induced uPA activity was detected in conditioned media in as little as 15 min, whereas PMA-induced uPA activity did not increase until 2 h. In addition to stimulating macrophage secretion of uPA, fucoidan bound uPA and had a small stimulatory affect on uPA activity. The binding does not interfere with the catalytic site on the B chain, or require the receptor binding or kringle domains on the A chain

Macrophage and foam cell release of matrix-bound growth factors. Role of plasminogen activation

We have determined whether macrophage derived-foam cells, a prominent component of the atherosclerotic lesion, express more urokinase-type plasminogen activator (uPA) and whether their ability to generate plasmin stimulates the release of matrix-bound growth factors. Steady state levels of uPA mRNA and both membrane and intracellular uPA activities were significantly increased in foam cells. When cultured on cell-derived matrices containing bound 125I-basic fibroblast growth factor (bFGF), both macrophage and foam cells released intact 125I-bFGF into their media. The release of 125I-bFGF by either cell was significantly enhanced in the presence of plasminogen. However, foam cells, which expressed more membrane uPA, released more 125I-bFGF than control cells. The release of matrix- bound bFGF was independent of heparanase activity, since neither macrophage nor foam cells degraded 35SO4-labeled heparan sulfate proteoglycans. In addition, media derived from foam cells cultured on cell-derived matrices in the presence of plasminogen had increased levels of transforming growth factor (TGF) β activity as compared to cells grown in the absence of plasminogen. In contrast, plasminogen had no effect on TGF-β activity recovered in the media of foam cells grown on plastic. Moreover, when macrophage were cultured on matrices containing bound 125I-TGF-β, the release of labeled TGF-β was increased in the presence of plasminogen. This is the first demonstration that foam cells can release two important growth regulators, bFGF and TGF-β, from the extracellular matrix, and provides a mechanism by which macrophage and foam cells can stimulate atherosclerotic lesion development

<i>CDKN2A</i> Deletions Define an Unfavorable Subgroup within the <i>MYD88/CD79B</i> (MCD) Subtype of Diffuse Large B-Cell Lymphoma (DLBCL) and Are Mutually Exclusive with <i>TP53</i> mutations

Abstract Background: Alterations (particularly biallelic deletions) of the tumor suppressor gene CDKN2A are frequent in the ultra-aggressive lymphoblastic (Quesnel et al, Blood 1995) and Burkitt lymphomas (Schmitz et al, Nature 2012). They also occur in DLBCL, and in prior studies they were associated with poor prognosis in conjunction with TP53 mutations (Jardin, Blood 2010). However, recent genomic classifications of DLBCL have noted frequent CDKN2A alterations in the MCD subtype (characterized by MYD88L265P and CD79 mutations; Wright et al, Cancer Cell 2020-LymphGen classifier). MCD tumors show propensity for extranodal invasion, immune evasion, and are enriched among relapsed/refractory DLBCL (Ollila et al, Blood 2021). There is an interest in targeting the MCD subgroup with novel treatment approaches, but prognostic factors specific to MCD DLBCL are uncertain. We examined the association between CDKN2A deletions and other mutations, genomic subtypes, and prognosis in DLBCL. Methods: We selected DLBCL cases submitted for next generation sequencing (NGS) as part of routine clinical care (FoundationOne Heme assay, Foundation Medicine, Inc., Cambridge, MA). All samples underwent central review by a board-certified pathologist. NGS was performed on hybridization-captured, adaptor ligation-based libraries in up to 405 cancer-related genes (Frampton et al, Nat Biotechnol, 2013), identifying clinically relevant base pair substitutions, indels, copy number alterations, and rearrangements. Co-occurrence/exclusivity was evaluated by odds ratios (OR) with P-values corrected for multiple testing using false discovery rate (FDR). Prognostic analysis was performed using publicly available data from the Haematological Malignancy Research Network (HMRN) study of 648 patients treated with RCHOP chemotherapy for DLBCL (Lacy et al, Blood 2020). Results: Among 165 patients with confirmed DLBCL, median age was 67 (interquartile range, 56-76), and 48% were women. Biopsies were from an extranodal site in 113 cases (68%). CDKN2A alterations were present in 42 samples (25%): most commonly biallelic deletions (N=34), short variant alterations (N=7), and 1 rearrangement. CDKN2A deletions were found in 28 (25%) of extranodal and 6 (12%) of nodal biopsies (Fisher's exact P=.06). MYC-IGH rearrangement was detected in 3 (7%) of tumors with CDKN2A deletions and 5 (4%) of those without them (P=.42), but BCL2-IGH rearrangement was rare in tumors with CDKN2A deletions (2% vs. 33%, respectively; P&amp;lt;0.001). Mutations in only 3 genes were statistically significantly associated with CDKN2A deletions: MYD88 (OR=12.6, Pcorr=3.9 x 10 -6), CD79B (OR=20.4, Pcorr =.00031) were highly co-occurring, whereas TP53 (OR=0.09, Pcorr=.0072) was highly mutually exclusive (Fig. A/B). Among tumors with CDKN2A deletions, 56% had mutations in MYD88, 32% in CD79B, and 32% in PIM1, but only 6% in TP53. Conversely, in DLBCL without CDKN2A deletions, TP53 mutations were present in 41%, while &amp;lt;10% had mutations in MYD88, CD79B, or PIM1. When studied using the LymphGen DLBCL classifier, CDKN2A deletions were present in 14 out of 16 MCD (88%), 2 out of 10 (20%) BN2, 18 out of 111 (16%) of unclassifiable tumors, and in no tumors classified as A53, EZB, or ST2 (Fig. C; P&amp;lt;.001 for MCD vs others). CDKN2A deletions were also specific to the hc-MCD subtype using our simplified hierarchical classifier developed for multi-gene NGS panels (Fig. D). In the HMRN data, CDKN2A deletions were observed in 10% of cases, significantly more often (34%) in the MYD88 cluster (corresponding to LymphGen MCD) than in other clusters (6.3%; P&amp;lt;.001). Conversely, TP53 alterations were significantly less frequent in the MYD88 cluster (7% vs 21% in others, P=.004). CDKN2A deletions were associated with significantly worse progression-free and overall survival (Fig. E/F) within the MYD88 cluster (independently of the International Prognostic Index), but not in others. Conclusions: CDKN2A deletions are specific to the MCD genomic subtype of DLBCL and indicate particularly poor prognosis within this class. Relative mutual exclusivity with TP53 mutations suggests that CDKN2A deletion may constitute an alternative, critical "hit" to a tumor suppressor gene in MCD DLBCL. Further research should examine the clinical relevance of CDKN2A deletions for refractoriness to standard therapy and its role in immune evasion that is characteristic of relapsed/refractory MCD DLBCL. Figure 1 Figure 1. Disclosures Olszewski: TG Therapeutics: Research Funding; PrecisionBio: Research Funding; Celldex Therapeutics: Research Funding; Acrotech Pharma: Research Funding; Genentech, Inc.: Research Funding; Genmab: Research Funding. Sharaf: Foundation Medicine: Current Employment. Marcus: Foundation Medicine: Current Employment. Albacker: Foundation Medicine: Current Employment. Vergilio: Foundation Medicine: Current Employment. </jats:sec