Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and ...size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain ...bears a mutation that increases Tfh cell number and heterozygous animals (Roquinsan/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquinsan/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2-inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4.sup.+ T-cell subsets, which are likely to contribute to the B-cell ...responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated. Methods Employing a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells. Results Both total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3.sup.+ expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1.sup.hi cells showed significantly more clustering than did FOXP3.sup.+. To investigate the association of infiltrating T-cells with lymphoma B-cells we employed Pearson correlation and Morisita-Horn index, statistical measures of interaction. We demonstrated that PD1.sup.hi cells were associated with proliferating B-cells and confirmed this by nearest neighbour analysis. Conclusions The unexpected architectural complexity of T-cell infiltration in marginal zone lymphoma, revealed in this study, further supports a key role for Tfh cells in driving proliferation of lymphoma B-cells. We demonstrate the feasibility of digital analysis of spatial architecture of T-cells within marginal zone lymphoma and future studies will be needed to determine the clinical importance of these observations. Keywords: Marginal zone lymphoma, Follicular helper T-cells, Spatial characteristics
Formation and fission of tubules from autolysosomes, endolysosomes, or phagolysosomes are required for lysosome reformation. However, the mechanisms governing these processes in these different ...lysosomal organelles are poorly understood. Thus, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIIIβ causes extensive lysosomal tubulation. Using super-resolution live-cell imaging, we show that Arf1-PI4KIIIβ positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes, and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIIIβ represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIIIβ positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIIIβ positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery.
Patients with peripheral T-cell lymphomas generally have poor clinical outcomes with conventional chemotherapy. Recent advances have demonstrated that a large subgroup of PTCL are derived from ...follicular helper (Tfh) T-cells. These cases show a characteristic pattern of gene expression, which includes high-level protein expression of interleukin-2-inducible kinase (ITK). ITK is a member of the TEC family of kinases and normally has essential functions in regulating T-cell receptor signalling and T-cell differentiation. Here we report a side-by-side comparison of four ITK inhibitors. We investigate effects on apoptosis, phosphorylation of signaling molecules, calcium flux and migration. In line with a specific mechanism of action ONO7790500 and BMS509744 did not inhibit MEK1/2 or AKT phosphorylation although other ITK inhibitors, ibrutinib and PF-06465469, did have this effect. Specific ITKi had modest effects on apoptosis alone but there was definite synergy with doxorubicin, pictilisib (PI3Ki) and idelalisib (PI3Kδi). ITKi repressed migration of Jurkat cells caused by CXCL12 and the CXCR4 antagonist, plerixafor enhanced this effect. Overall ITKi may have several mechanisms of action that will be therapeutically useful in PTCL including reduction in survival and perturbation of trafficking.
The IKK‐related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll‐like receptors to regulate NF‐κB signalling. We investigated ...the expression and function of IKKε and TBK1, in diffuse large B‐cell lymphoma (DLBCL). DLBCL cell lines and patient‐derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non‐germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL‐10 production from Ly10 and repressed NF‐κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF‐κB signalling in lymphoma.
Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain ...bears a mutation that increases Tfh cell number and heterozygous animals (Roquin.sup.san/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquin.sup.san/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2-inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Peripheral T cell lymphoma (PTCL) has a poor prognosis and there is a need for new treatments.
The sanroque mouse bears a mutation in the E3 ubiquitin ligase, Roquin, which leads to T-cell activation ...and autoimmunity in homozygous animals. In heterozygous mice (Roquinsan/+) T-cell lymphomas with histological similarity to human angioimmunoblastic lymphoma (AITL), the commonest subtype of PTCL, develop in 40 to 50% of animals by 6 - 12 months of age.
In order to test the feasibility of using sanroque mice for pre-clinical work we carried out MRI scans of the axillary and inguinal regions before and after receiving 200mg/kg cyclophosphamide as an intra-peritoneal injection. After 28-days the MRI scans were repeated and lymph nodes, spleens and livers collected.
MRI scanning was performed on a 9.4T Agilent scanner (Agilent Technologies, Santa Claire, CA, USA) with a 310mm bore diameter and 6cm inner-diameter gradient (1000mT/m maximum gradient strength). A 4cm millipede RF coil was used for RF transmission and reception. T2-weighted images were acquired using a respiratory-gated fast spin echo (FSE) sequence with TR/TE=3000/40ms, 40x40mm field of view (256x256 matrix), 32x0.8mm slices and two signal averages.
Enlarged lymph nodes were identified on the pre-treatment MRI scan and compared to the equivalent lymph node following treatment (Figure 1A). There was a mean 94% reduction in lymph node size following cyclophosphamide treatment. This demonstrated that the enlarged lymph nodes were chemosensitive and that MRI scanning could be used to monitor the lymph node size in response to treatment.
Interleukin-2-inducible kinase (ITK) is expressed and has essential functions in regulating normal T-cell proliferation and differentiation. ITK is also expressed in AITL and might be a therapeutic target but there is no pre-clinical data. The small molecule BTK/ITK inhibitor, ibrutinib, is effective and well tolerated in clinical practice in some B-cell lymphomas and ibrutinib is active against mouse ITK.
In order to assess the effect of ibrutinib on (Roquinsan/+) T-cell lymphoma we identified twelve mice with palpable lymph nodes. Following an MRI scan, oral treatment with ibrutinib (25mg/kg in 1% (2-Hydroxypropyl)-β-cyclodextrin (2HPBD)) or equivalent volume of vehicle (1% 2HPBD) was given daily by gavage. Five mice were treated with ibrutinib for 28 days and three for reduced time periods (7 days, 14 days and 21 days). Four mice were treated with vehicle for 28 days. Following cessation of therapy a further MRI scan was performed prior to harvesting of tissues, including liver, spleen and lymph nodes. MRI images were analysed to determine the change in volume of lymph nodes during the treatment period.
Responses to ibrutinib were mixed (Figure 1B & 1C): of those that received 28 days treatment 5/8 animals showed reduction in tumour size of between 10 and 86% whilst those that received shorter durations of treatment showed reductions of 8 and 11%. 3/8 ibrutinib treated animals showed increases in tumour size of 9 and 85% (mice treated for 28 days) whilst one animal treated for 7 days showed an increase of 117%. 3/4 vehicle treated animals showed stable or increasing lymphadenopathy whilst one mouse showed spontaneous tumour regression.
In order to analyze the effects of ibrutinib at the cellular level we established flow cytometry protocols to determine cellular composition. Previous reports show that the majority of cells within sanroque enlarged lymph nodes are B-cells with a minority (1 to 2%) being malignant T-cells. This is similar to the pattern observed in human Tfh lymphoma. Lymph nodes were harvested following the post-treatment MRI scan and cell suspensions produced. The proportions of CD4+, CD8+ and B220+ lymphocytes were assessed. To determine the Tfh cell population CD4 positive lymphocytes were gated and the proportion of CXCR5hiPD1hi cells assessed. We did not observe any significant differences between responding and non-responding mice (Figure 1D) or treated and untreated mice.
We have developed an MRI protocol to assess the response of a T-cell lymphoma model to novel agents to facilitate pre-clinical studies. We interpret our results as showing that ibrutinib is effective as a single agent in a proportion of the spontaneous T-cell lymphomas generated in the Roquinsan/+ model of Tfh lymphoma but further work is required to understand the differences between responder and non-responder tumours.
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No relevant conflicts of interest to declare.
Abstract
Genetic lesions are central contributors to cancer development and progression, and understanding the sources of these lesions will provide a better understanding of the mechanisms of ...carcinogenesis. Our previous work showed that the uncharacterized murine Fam72a gene promotes mutagenic DNA repair during antibody maturation by causing the degradation of Uracil DNA glycosylase 2 (UNG2), a component of the base excision repair (BER) system. Humans encode four almost identical paralogues of FAM72 called FAM72A-D, which are absent in all other mammalian genomes, including those of non-human primates. The role of these four paralogues in human biology is unknown. Intriguingly, a previous report showed that FAM72A is over-expressed in many cancers. Since FAM72A promotes mutagenesis by degradation of UNG2, we hypothesized that overexpression of the FAM72 gene family might promote cancer development and progression. Here we show that FAM72A-D is overexpressed in many human cancers and inversely correlates with UNG2 protein levels in tumorigenic tissue. However, FAM72A but not FAM72B-D causes degradation of UNG2. Consistent with this effect, FAM72 expression correlates with a higher mutation load in many tumor types. Since genetic mutations are associated with cancer development and progression, we tested whether FAM72 expression is associated with disease outcomes. Indeed, we found that high FAM72 expression was associated with poorer survival in several cancers. To directly test if Fam72a is sufficient to promote cancer, we generated transgenic mice that overexpress Fam72a in multiple tissues. We observed that Fam72a overexpression promotes increased colonic polyps in the Apcmin background compared to controls. These data show that the novel FAM72 gene family are drivers of cancer development in both mice and humans and advances our understanding of the underlying molecular mechanisms that precipitate cancer development and progression. This work is supported by a grant from the CIHR (PJT-180269).
Citation Format: Yuqing Feng, Bhupesh Thakur, Jeffery Bruce, Sami Mamand, Melika Shirdarreh, Matthew Wong, Jennifer Silvester, Ming Han, Mohammad Kashem, Amin Zia, David Cescon, Tervor Pugh, Rossanna C. Pezo, Alberto Martin. The FAM72 gene family promotes cancer development by disabling the base excision repair system abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1217.
The mutational landscape of peripheral T-cell lymphoma (PTCL) is being revealed through sequencing of lymph node samples, but there has been little work on the mutational load that is present in ...cell-free DNA (cfDNA) from plasma. We report targeted sequencing of cfDNA from PTCL patients to demonstrate c.50G>T (p.Gly17Val) in RHOA as previously described in angioimmunoblastic T-cell lymphoma (AITL) and a group of PTCL not otherwise specified (NOS) but also detect novel mutations at c.73A>G (p.Phe25Leu) and c.48A>T (p.Cys16*) of exon 2, which were confirmed by Sanger sequencing. In a group of AITL and PTCL-NOS analyzed by droplet digital polymerase chain reaction, 63% (12/19) showed c.50G>T (p.Gly17Val), 53% (10/19) c.73A>G (p.Phe25Leu), and 37% (7/19) c.48A>T (pCys16*). Sequencing of lymph node tissue in 3 out of 9 cases confirmed the presence of c.73A>G (p.Phe25Leu). Inspection of individual sequencing reads from individual patients showed that a single RHOA allele could contain >1 mutation, suggesting haplotypes of mutations at RHOA. Serial sampling showed changes to RHOA mutational frequency with treatment and the apparent occurrence of clones bearing specific haplotypes associated with relapse. Therefore, sequencing of RHOA from cfDNA has revealed new mutations and haplotypes. The clinical significance of these findings will need to be explored in clinical trials, but liquid biopsy might have potential for guiding treatment decisions in PTCL.
•Multiple RHOA mutations can combine in haplotypes and are detectable in cfDNA from plasma of patients with PTCL.•Serial cfDNA monitoring of RHOA mutations could allow molecular tracking of disease response in PTCL patients undergoing treatment.
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