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Understanding the molecular subtype of a cancer is becoming an importance part of the diagnostic process as it helps a doctor better understand a patient’s prognosis, determine the best course of action for treatment and helps researchers devise new, more-efficient, precision therapies.
Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O2) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O2). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.
In mammalian cells, cell cycle entry occurs in response to the correct stimuli and is promoted by the transcriptional activity of E2F family members. E2F proteins regulate the transcription of S phase cyclins and genes required for DNA replication, DNA repair, and apoptosis. The activity of E2F1, the archetypal and most heavily studied E2F family member, is tightly controlled by the DNA damage checkpoints to modulate cell cycle progression and initiate programmed cell death, when required. Altered tumor suppressor and oncogenic signaling pathways often result in direct or indirect interference with E2F1 regulation to ensure higher rates of cell proliferation independently of external cues. Despite a clear link between dysregulated E2F1 activity and cancer progression, literature on the contribution of E2F1 to DNA replication stress phenotypes is somewhat scarce. This review discusses how dysfunctional tumor suppressor and oncogenic signaling pathways promote the disruption of E2F1 transcription and hence of its transcriptional targets, and how such events have the potential to drive DNA replication stress. In addition to the involvement of E2F1 upstream of DNA replication stress, this manuscript also considers the role of E2F1 as a downstream effector of the response to this type of cellular stress. Lastly, the review introduces some reflections on how E2F1 activity is integrated with checkpoint control through post-translational regulation, and proposes an exploitable tumor weakness based on this axis.
Treatment with ionizing radiation (IR) remains the cornerstone of therapy for multiple cancer types, including disseminated and aggressive diseases in the palliative setting. Radiotherapy efficacy could be improved in combination with drugs that regulate the ubiquitin-proteasome system (UPS), many of which are currently being tested in clinical trials. The UPS operates through the covalent attachment of ATP-activated ubiquitin molecules onto substrates following the transfer of ubiquitin from an E1, to an E2, and then to the substrate via an E3 enzyme. The specificity of ubiquitin ligation is dictated by E3 ligases, which select substrates to be ubiquitylated. Among the E3s, cullin ring ubiquitin ligases (CRLs) represent prototypical multi-subunit E3s, which use the cullin subunit as a central assembling scaffold. CRLs have crucial roles in controlling the cell cycle, hypoxia signaling, reactive oxygen species clearance and DNA repair; pivotal factors regulating the cancer and normal tissue response to IR. Here, we summarize the findings on the involvement of CRLs in the response of cancer cells to IR, and we discuss the therapeutic approaches to target the CRLs which could be exploited in the clinic.
Despite recent advances in our understanding of the disease, glioblastoma (GB) continues to have limited treatment options and carries a dismal prognosis for patients. Efforts to stratify this heterogeneous malignancy using molecular classifiers identified frequent alterations in targetable proteins belonging to several pathways including the receptor tyrosine kinase (RTK) and mitogen-activated protein kinase (MAPK) signalling pathways. However, these findings have failed to improve clinical outcomes for patients. In almost all cases, GB becomes refractory to standard-of-care therapy, and recent evidence suggests that disease recurrence may be associated with a subpopulation of cells known as glioma stem cells (GSCs). Therefore, there remains a significant unmet need for novel therapeutic strategies. E3 ubiquitin ligases are a family of >700 proteins that conjugate ubiquitin to target proteins, resulting in an array of cellular responses, including DNA repair, pro-survival signalling and protein degradation. Ubiquitin modifications on target proteins are diverse, ranging from mono-ubiquitination through to the formation of polyubiquitin chains and mixed chains. The specificity in substrate tagging and chain elongation is dictated by E3 ubiquitin ligases, which have essential regulatory roles in multiple aspects of brain cancer pathogenesis. In this review, we begin by briefly summarising the histological and molecular classification of GB. We comprehensively describe the roles of E3 ubiquitin ligases in RTK and MAPK, as well as other, commonly altered, oncogenic and tumour suppressive signalling pathways in GB. We also describe the role of E3 ligases in maintaining glioma stem cell populations and their function in promoting resistance to ionizing radiation (IR) and chemotherapy. Finally, we consider how our knowledge of E3 ligase biology may be used for future therapeutic interventions in GB, including the use of blood-brain barrier permeable proteolysis targeting chimeras (PROTACs).
Ongoing repair of migration-coupled DNA damage allows planarian adult stem cells to reach wound sites.
Mechanical stress during cell migration may be a previously unappreciated source of genome instability, but the extent to which this happens in any animal in vivo remains unknown. We consider an in vivo system where the adult stem cells of planarian flatworms are required to migrate to a distal wound site. We observe a relationship between adult stem cell migration and ongoing DNA damage and repair during tissue regeneration. Migrating planarian stem cells undergo changes in nuclear shape and exhibit increased levels of DNA damage. Increased DNA damage levels reduce once stem cells reach the wound site. Stem cells in which DNA damage is induced prior to wounding take longer to initiate migration and migrating stem cell populations are more sensitive to further DNA damage than stationary stem cells. RNAi mediated knockdown of DNA repair pathway components blocks normal stem cell migration, confirming that active DNA repair pathways are required to allow successful migration to a distal wound site. Together these findings provide evidence that levels of Migration-Coupled-DNA-Damage (MCDD) are significant in adult stem cells and that ongoing migration requires DNA repair mechanisms. Our findings reveal that migration of normal stem cells in vivo represent an unappreciated source of damage, that could be a significant source of mutations in animals during development or during long term tissue homeostasis.
We introduce Region of Interest Contrast Enhancement (RICE) to identify focal densities in mammograms. It aims to help radiologists: 1) enhancing the contrast of mammographic images; and 2) detecting regions of interest (such as focal densities) that are candidate masses potentially masked behind dense parenchyma. Cancer masking is an unsolved issue, particularly in breast density categories BI-RADS C and D. RICE suppresses normal breast parenchyma in order to highlight focal densities. Unlike methods that enhance mammograms by modifying the dynamic range of an image; RICE relies on the actual tissue composition of the breast. It segments Volumetric Breast Density (VBD) maps into smaller regions and then applies a recursive mechanism to estimate the 'neighbourhood' for each segment. The method then subtracts and updates the neighbourhood, or the encompassing tissue, from each piecewise constant component of the breast image. This not only enhances the appearance of a candidate mass but also helps in estimating the mass density. In extensive experiments, RICE enhances focal densities in all breast density types including the most challenging category BI-RADS D. Suitably adapted, RICE can be used as a precursor to any computer-aided diagnostics and detection system.
Oncolytic herpesvirus expressing PD-L1 BiTE for cancer therapy: exploiting tumor immune suppression as an opportunity for targeted immunotherapy.
BACKGROUND: Programmed death-ligand 1 (PD-L1) is an important immune checkpoint protein that can be regarded as a pan-cancer antigen expressed by multiple different cell types within the tumor. While antagonizing PD-L1 is well known to relieve PD-1/PD-L1-mediated T cell suppression, here we have combined this approach with an immunotherapy strategy to target T cell cytotoxicity directly toward PD-L1-expressing cells. We developed a bi-specific T cell engager (BiTE) crosslinking PD-L1 and CD3ε and demonstrated targeted cytotoxicity using a clinically relevant patient-derived ascites model. This approach represents an immunological 'volte-face' whereby a tumor immunological defense mechanism can be instantly transformed into an Achilles' heel for targeted immunotherapy. METHODS: The PD-L1 targeting BiTE comprises an anti-PD-L1 single-chain variable fragment (scFv) or nanobody (NB) domain and an anti-CD3 scFv domain in a tandem repeat. The ability to activate T cell cytotoxicity toward PD-L1-expressing cells was established using human carcinoma cells and PD-L1-expressing human ('M2') macrophages in the presence of autologous T cells. Furthermore, we armed oncolytic herpes simplex virus-1 (oHSV-1) with PD-L1 BiTE and demonstrated successful delivery and targeted cytotoxicity in unpurified cultures of malignant ascites derived from different cancer patients. RESULTS: PD-L1 BiTE crosslinks PD-L1-positive cells and CD3ε on T cells in a 'pseudo-synapse' and triggers T cell activation and release of proinflammatory cytokines such as interferon-gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10) and tumour necrosis factor-α (TNF-α). Activation of endogenous T cells within ascites samples led to significant lysis of tumor cells and M2-like macrophages (CD11b+CD64+ and CD206+/CD163+). The survival of CD3+ T cells (which can also express PD-L1) was unaffected. Intriguingly, ascites fluid that appeared particularly immunosuppressive led to higher expression of PD-L1 on tumor cells, resulting in improved BiTE-mediated T cell activation. CONCLUSIONS: The study reveals that PD-L1 BiTE is an effective immunotherapeutic approach to kill PD-L1-positive tumor cells and macrophages while leaving T cells unharmed. This approach activates endogenous T cells within malignant ascites, generates a proinflammatory response and eliminates cells promoting tumor progression. Using an oncolytic virus for local expression of PD-L1 BiTE also prevents 'on-target off-tumor' systemic toxicities and harnesses immunosuppressive protumor conditions to augment immunotherapy in immunologically 'cold' clinical cancers.
The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.
Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
An electron beam of very high energy (50-250 MeV) can potentially produce a more favourable radiotherapy dose distribution compared to a state-of-the-art photon based radiotherapy technique. To produce an electron beam of sufficiently high energy to allow for a long penetration depth (several cm), very large accelerating structures are needed when using conventional radio-frequency technology, which may not be possible due to economical or spatial constraints. In this paper, we show transport and focusing of laser wakefield accelerated electron beams with a maximum energy of 160 MeV using electromagnetic quadrupole magnets in a point-to-point imaging configuration, yielding a spatial uncertainty of less than 0.1 mm, a total charge variation below [Formula: see text] and a focal spot of [Formula: see text]. The electron beam was focused to control the depth dose distribution and to improve the dose conformality inside a phantom of cast acrylic slabs and radiochromic film. The phantom was irradiated from 36 different angles to obtain a dose distribution mimicking a stereotactic radiotherapy treatment, with a peak fractional dose of 2.72 Gy and a total maximum dose of 65 Gy. This was achieved with realistic constraints, including 23 cm of propagation through air before any dose deposition in the phantom.
Ultra-high-dose-rate FLASH and Conventional-Dose-Rate Irradiation Differentially Affect Human Acute Lymphoblastic Leukemia and Normal Hematopoiesis.
PURPOSE: Ultra-high-dose-rate FLASH radiation therapy has been shown to minimize side effects of irradiation in various organs while keeping antitumor efficacy. This property, called the FLASH effect, has caused enthusiasm in the radiation oncology community because it opens opportunities for safe dose escalation and improved radiation therapy outcome. Here, we investigated the impact of ultra-high-dose-rate FLASH versus conventional-dose-rate (CONV) total body irradiation (TBI) on humanized models of T-cell acute lymphoblastic leukemia (T-ALL) and normal human hematopoiesis. METHODS AND MATERIALS: We optimized the geometry of irradiation to ensure reproducible and homogeneous procedures using eRT6/Oriatron. Three T-ALL patient-derived xenografts and hematopoietic stem/progenitor cells (HSPCs) and CD34+ cells isolated from umbilical cord blood were transplanted into immunocompromised mice, together or separately. After reconstitution, mice received 4 Gy FLASH and CONV-TBI, and tumor growth and normal hematopoiesis were studied. A retrospective study of clinical and gene-profiling data previously obtained on the 3 T-ALL patient-derived xenografts was performed. RESULTS: FLASH-TBI was more efficient than CONV-TBI in controlling the propagation of 2 cases of T-ALL, whereas the third case of T-ALL was more responsive to CONV-TBI. The 2 FLASH-sensitive cases of T-ALL had similar genetic abnormalities, and a putative susceptibility imprint to FLASH-RT was found. In addition, FLASH-TBI was able to preserve some HSPC/CD34+ cell potential. Interestingly, when HSPC and T-ALL were present in the same animals, FLASH-TBI could control tumor development in most (3 of 4) of the secondary grafted animals, whereas among the mice receiving CONV-TBI, treated cells died with high leukemia infiltration. CONCLUSIONS: Compared with CONV-TBI, FLASH-TBI reduced functional damage to human blood stem cells and had a therapeutic effect on human T-ALL with a common genetic and genomic profile. The validity of the defined susceptibility imprint needs to be investigated further; however, to our knowledge, the present findings are the first to show benefits of FLASH-TBI on human hematopoiesis and leukemia treatment.
Hypofractionated FLASH-RT as an Effective Treatment against Glioblastoma that Reduces Neurocognitive Side Effects in Mice.
PURPOSE: Recent data have shown that single-fraction irradiation delivered to the whole brain in less than tenths of a second using FLASH radiotherapy (FLASH-RT), does not elicit neurocognitive deficits in mice. This observation has important clinical implications for the management of invasive and treatment-resistant brain tumors that involves relatively large irradiation volumes with high cytotoxic doses. EXPERIMENTAL DESIGN: Therefore, we aimed at simultaneously investigating the antitumor efficacy and neuroprotective benefits of FLASH-RT 1-month after exposure, using a well-characterized murine orthotopic glioblastoma model. As fractionated regimens of radiotherapy are the standard of care for glioblastoma treatment, we incorporated dose fractionation to simultaneously validate the neuroprotective effects and optimized tumor treatments with FLASH-RT. RESULTS: The capability of FLASH-RT to minimize the induction of radiation-induced brain toxicities has been attributed to the reduction of reactive oxygen species, casting some concern that this might translate to a possible loss of antitumor efficacy. Our study shows that FLASH and CONV-RT are isoefficient in delaying glioblastoma growth for all tested regimens. Furthermore, only FLASH-RT was found to significantly spare radiation-induced cognitive deficits in learning and memory in tumor-bearing animals after the delivery of large neurotoxic single dose or hypofractionated regimens. CONCLUSIONS: The present results show that FLASH-RT delivered with hypofractionated regimens is able to spare the normal brain from radiation-induced toxicities without compromising tumor cure. This exciting capability provides an initial framework for future clinical applications of FLASH-RT.See related commentary by Huang and Mendonca, p. 662.
INTRODUCTION: The α-chain variant Hb Q-India (c.193G>C) is caused by a point mutation GAC→CAC at codon 64 of the α1 globin gene and is clinically silent. Point mutations can be diagnosed easily by many simple polymerase chain reaction (PCR) techniques including PCR-restriction digest, but for Hb Q-India the restriction digest has never been described. In this work we aimed to develop a restriction enzyme digestion assay for DNA diagnosis of Hb Q-India, in order to increase the panel of restriction enzymes used in DNA diagnosis of haemoglobinopathies and also as a simple cheap alternative to the ARMS-PCR method. METHODS: A restriction enzyme digestion assay was designed for diagnosis of Hb Q-India using the restriction enzyme EaeI enzyme as the Hb Q-India mutation abolishes the recognition site of this enzyme. Patients were screened for an abnormal haemoglobin by high performance liquid chromatography (HPLC) and those had an abnormal peak with a retention time between 4.7 and 4.8 minutes were selected for diagnosis at the molecular level. The α1 globin gene was amplified in 12 cases with a presumed diagnosis of Hb Q-India by HPLC and isoelectric focusing (IEF), and the amplified products were subjected to the EaeI digestion. RESULTS: All the 12 cases were diagnosed positive (100%) for Hb Q-India by the EaeI restriction enzyme digest. They were heterozygotes for the mutation. CONCLUSION: EaeI restriction enzyme digestion can be used as a simple and robust alternative method to ARMS-PCR for DNA diagnosis of Hb Q-India. The EaeI restriction enzyme can be added to the panel of restriction enzymes used in the DNA diagnosis of the abnormal Hb variants. Concomitant use of HPLC and IEF can be used efficiently for presumed diagnosis of this rare variant.
Ofatumumab retreatment and maintenance in fludarabine-refractory chronic lymphocytic leukaemia patients.
There are limited data on retreatment with monoclonal antibodies (mAb) in patients with chronic lymphocytic leukaemia (CLL). In a pivotal study, ofatumumab (human anti-CD20 mAb) monotherapy demonstrated a 47% objective response rate (ORR) in fludarabine refractory CLL patients. From this study, a subset of 29 patients who had at least stable disease and then progressed were retreated with eight weekly ofatumumab infusions (induction treatment period), followed by monthly infusions for up to 2 years (maintenance treatment period). The ORR after 8 weeks of induction retreatment was 45% and 24% had continued disease control after maintenance at 52 weeks. Efficacy and safety of the retreated patients were compared with their initial results in the pivotal study. Response duration was 24.1 months vs. 6.8 months; time to next therapy was 14.8 months vs. 12.3 months; and progression-free survival was 7.4 months vs. 7.9 months (medians). Upon retreatment, 72% had infusion reactions, mostly Grade 1-2. Three patients had fatal infections. In summary, ofatumumab retreatment and maintenance therapy was feasible in patients with heavily pretreated CLL and appeared to result in more durable disease control than initial ofatumumab treatment in this subset of patients who may have a more favourable disease profile.