The anti-cancer agent APR-246 can activate several programmed cell death processes to kill malignant cells.

Mutant TP53 proteins are thought to drive the development and sustained expansion of cancers at least in part through the loss of the wild-type (wt) TP53 tumour suppressive functions. Therefore, compounds that can restore wt TP53 functions in mutant TP53 proteins are expected to inhibit the expansion of tumours expressing mutant TP53. APR-246 has been reported to exert such effects in malignant cells and is currently undergoing clinical trials in several cancer types. However, there is evidence that APR-246 may also kill malignant cells that do not express mutant TP53. To support the clinical development of APR-246 it is important to understand its mechanism(s) of action. By establishing isogenic background tumour cell lines with different TP53/TRP53 states, we found that APR-246 can kill malignant cells irrespective of their TP53/TRP53 status. Accordingly, RNAseq analysis revealed that treatment with APR-246 induces expression of the same gene set in Eµ-Myc mouse lymphoma cells of all four possible TRP53 states, wt, wt alongside mutant, knockout and knockout alongside mutant. We found that depending on the type of cancer cell and the concentration of APR-246 used, this compound can kill malignant cells through induction of various programmed cell death pathways, including apoptosis, necroptosis and ferroptosis. The sensitivity of non-transformed cells to APR-246 also depended on the cell type. These findings reveal that the clinical testing of APR-246 should not be limited to cancers expressing mutant TP53 but expanded to cancers that express wt TP53 or are TP53-deficient.

Proinflammatory microenvironment promotes lymphoma progression in mice with high megakaryocyte and TPO levels.

Platelets have been shown to enhance the survival of lymphoma cell lines. However, it remains unclear whether they play a role in lymphoma. Here, we investigated the potential role of platelets and/or megakaryocytes in the progression of Eµ-myc lymphoma. Eµ-myc tumor cells were transplanted into recipient wild-type (WT) control, Mpl-/-, or TpoTg mice, which exhibited normal, low, and high platelet and megakaryocyte counts, respectively. TpoTg mice that underwent transplantation exhibited enhanced lymphoma progression with increased white blood cell (WBC) counts, spleen and lymph node weights, and enhanced liver infiltration when compared with WT mice. Conversely, tumor-bearing Mpl-/- mice had reduced WBC counts, lymph node weights, and less liver infiltration than WT mice. Using an Mpl-deficient thrombocytopenic immunocompromised mouse model, our results were confirmed using the human non-Hodgkin lymphoma GRANTA cell line. Although we found that platelets and platelet-released molecules supported Eµ-myc tumor cell survival in vitro, pharmacological inhibition of platelet function or anticoagulation in WT mice transplanted with Eµ-myc did not improve disease outcome. Furthermore, transient platelet depletion or sustained Bcl-xL-dependent thrombocytopenia did not alter lymphoma progression. Cytokine analysis of the bone marrow fluid microenvironment revealed increased levels of the proinflammatory molecule interleukin 1 in TpoTg mice, whereas these levels were lower in Mpl-/- mice. Moreover, RNA sequencing of blood-resident Eµ-myc lymphoma cells from TpoTg and WT mice after tumor transplantation revealed the upregulation of hallmark gene sets associated with an inflammatory response in TpoTg mice. We propose that the proinflammatory microenvironment in TpoTg mice promotes lymphoma progression.

BH3 mimetic drugs cooperate with Temozolomide, JQ1 and inducers of ferroptosis in killing glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

Molecular mechanisms of cell death in neurological diseases.

Tightly orchestrated programmed cell death (PCD) signalling events occur during normal neuronal development in a spatially and temporally restricted manner to establish the neural architecture and shaping the CNS. Abnormalities in PCD signalling cascades, such as apoptosis, necroptosis, pyroptosis, ferroptosis, and cell death associated with autophagy as well as in unprogrammed necrosis can be observed in the pathogenesis of various neurological diseases. These cell deaths can be activated in response to various forms of cellular stress (exerted by intracellular or extracellular stimuli) and inflammatory processes. Aberrant activation of PCD pathways is a common feature in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease, resulting in unwanted loss of neuronal cells and function. Conversely, inactivation of PCD is thought to contribute to the development of brain cancers and to impact their response to therapy. For many neurodegenerative diseases and brain cancers current treatment strategies have only modest effect, engendering the need for investigations into the origins of these diseases. With many diseases of the brain displaying aberrations in PCD pathways, it appears that agents that can either inhibit or induce PCD may be critical components of future therapeutic strategies. The development of such therapies will have to be guided by preclinical studies in animal models that faithfully mimic the human disease. In this review, we briefly describe PCD and unprogrammed cell death processes and the roles they play in contributing to neurodegenerative diseases or tumorigenesis in the brain. We also discuss the interplay between distinct cell death signalling cascades and disease pathogenesis and describe pharmacological agents targeting key players in the cell death signalling pathways that have progressed through to clinical trials.

CuII(atsm) Attenuates Neuroinflammation.

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer's disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation. Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex CuII(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro. Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of CuII(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). CuII(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes. Conclusion: The beneficial effects of CuII(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.

Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato)copper(II) Complexes on Cellular Copper Uptake, Copper-Mediated ATP7A Trafficking, and P-Glycoprotein Mediated Efflux.

Bis(thiosemicarbazonato)copper(II) complexes are of interest as potential therapeutics for cancer and neurodegenerative diseases as well as imaging agents for positron emission tomography (PET). The cellular uptake of six bis(thiosemcarbazonato)copper(II)complexes derived from glyoxal, with different functional groups Cu(gtsx) where x = different functional groups, was investigated in SKOV-3, HEK293, and HEK293 P-gp cell lines. Treatment of the cells with the copper complexes increased intracellular copper and increased levels of p-ERK due to activation of the Ras-Raf-MEK-ERK pathway. Treatment of SKOV-3 cells with low concentrations (µM) of two of the copper complexes led to trafficking of the endogenous copper transporter ATP7A from the Golgi network to the cell membrane. Experiments in HEK293 and HEK293-P-gp cells suggest that Cu(gtsm) and Cu(gtse) are substrates for the P-gp efflux protein but the complex with a pyrrolidine functional group, Cu(gtspyr), is not. A PET experiment in mice showed that [64Cu]Cu(gtspyr) has reasonable brain uptake but high liver uptake.

Altered B-lymphopoiesis in mice with deregulated thrombopoietin signaling.

Thrombopoietin (TPO) is the master cytokine regulator of megakaryopoiesis. In addition to regulation of megakaryocyte and platelet number, TPO is important for maintaining proper hematopoietic stem cell (HSC) function. It was previously shown that a number of lymphoid genes were upregulated in HSCs from Tpo -/- mice. We investigated if absent or enhanced TPO signaling would influence normal B-lymphopoiesis. Absent TPO signaling in Mpl -/- mice led to enrichment of a common lymphoid progenitor (CLP) signature in multipotential lineage-negative Sca-1+c-Kit+ (LSK) cells and an increase in CLP formation. Moreover, Mpl -/- mice exhibited increased numbers of PreB2 and immature B-cells in bone marrow and spleen, with an increased proportion of B-lymphoid cells in the G1 phase of the cell cycle. Conversely, elevated TPO signaling in Tpo Tg mice was associated with reduced B-lymphopoiesis. Although at steady state, peripheral blood lymphocyte counts were normal in both models, Mpl -/- Eµ-myc mice showed an enhanced preneoplastic phase with increased numbers of splenic PreB2 and immature B-cells, a reduced quiescent fraction, and augmented blood lymphocyte counts. Thus, although Mpl is not expressed on lymphoid cells, TPO signaling may indirectly influence B-lymphopoiesis and the preneoplastic state in Myc-driven B-cell lymphomagenesis by lineage priming in multipotential progenitor cells.

Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways.

BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress.

Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219-414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.

Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim.

The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy.