Alzheimer's Disease (AD) is characterized by cognitive impairment, progressive neurodegeneration, and formation of amyloid-[Beta] (A[Beta])-containing plaques. These neuropathological features are accompanied by deregulation of signaling cascades such as the cyclin-dependent kinase- 5 (CDK5) pathway. Recent studies have revealed that neurodegeneration in AD is also associated with alterations in hippocampal neurogenesis, which may play a critical role in cognitive impairments and memory loss. The main objectives of this dissertation were to investigate the cellular mechanisms of defective hippocampal neurogenesis in an animal model of AD, and to examine the role of A[beta] and CDK5, downstream targets, and other potential candidate regulators in the mechanisms of defective neurogenesis in AD. To address these aims, in vitro and in vivo studies designed to inhibit CDK5 activity using pharmacological and genetic approaches demonstrated that CDK5 is critical for hippocampal neurogenesis. In an in vitro model of CDK5 hyperactivation, and in the hippocampus of amyloid precursor protein (APP) transgenic (tg) mice, markers of neurogenesis and neurite outgrowth were reduced. I identified the CDK5 substrate collapsin response mediator protein-2 (CRMP2) as a critical regulator of the effects of hyperactive CDK5 on neuronal maturation in AD. CDK5-mediated hyperphosphorylation of CRMP2 contributed to reduced neurite outgrowth by disrupting microtubule polymerization, and this effect could be rescued by down-regulating CDK5 or blocking CRMP2 phoshorylation. In addition to the role of the CDK5 pathway in neurogenesis in AD, other factors may be involved. In this context, defective hippocampal neurogenesis in AD patients and in APP tg mice was accompanied by increased expression levels of bone morphogenetic protein-6 (BMP6). In vitro studies in NPCs showed that Aβ increased BMP6 levels, and that BMP6 treatment reduced proliferation, supporting a role for BMP6 in hippocampal neurogenesis in AD. Lastly, we found that treatment of APP tg mice with a neurotrophic compound (Cerebrolysin) rescued hippocampal neurogenesis, and this effect was associated with down-regulation of the CDK5 signaling pathway. Taken together, these studies demonstrate that the CDK5 and BMP signaling cascades play important roles in adult hippocampal neurogenesis. Future therapeutic approaches could target these molecular pathways to rescue defective neurogenesis in AD

Neurodegenerative disorders of the aging population affect over 5 million people in the US and Europe alone. The common feature is the progressive accumulation of misfolded proteins with the formation of toxic oligomers. Alzheimer’s disease (AD) is characterized by cognitive impairment, progressive degeneration of neuronal populations in the neocortex and limbic system, and formation of amyloid plaques and neurofibrillary tangles. Amyloid-β (Aβ) is the product of proteolysis of amyloid precursor protein (APP) by β and γ-secretase enzymes. The neurodegenerative process in AD initiates with axonal and synaptic damage and is associated with progressive accumulation of toxic Aβ oligomers in the intracellular and extracellular space. In addition, neurodegeneration in AD is associated with alterations in neurogenesis. Aβ accumulation is the consequence of an altered balance between protein synthesis, aggregation rate, and clearance. Identification of genetic mutations in APP associated with familial forms of AD and gene polymorphisms associated with the more common sporadic variants of AD has led to the development of transgenic (tg) and knock out rodents as well as viral vector driven models of AD. While APP tg murine models with mutations in the N- and C-terminal flanking regions of Aβ are characterized by increased Aβ production with plaque formation, mutations in the mid-segment of Aβ result in increased formation of oligomers, and mutations toward the C-terminus (E22Q) segment results in amyloid angiopathy. Similar to AD, in APP tg models bearing familial mutations, formation of Aβ oligomers results in defective plasticity in the perforant pathway, selective neuronal degeneration, and alterations in neurogenesis. Promising results have been obtained utilizing APP tg models of AD to develop therapies including the use of β- and γ-secretase inhibitors, immunization, and stimulating neurogenesis.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common causes of dementia and movement disorders in the elderly. While progressive accumulation of oligomeric amyloid-β protein (Aβ) has been identified as one of the central toxic events in AD leading to synaptic dysfunction, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers has been linked to PD. Most of the studies in AD have been focused on investigating the role of Aβ and Tau; however, recent studies suggest that α-syn might also play a role in the pathogenesis of AD. For example, fragments of α-syn can associate with amyloid plaques and Aβ promotes the aggregation of α-syn in vivo and worsens the deficits in α-syn tg mice. Moreover, α-syn has also been shown to accumulate in limbic regions in AD, Down’s syndrome, and familial AD cases. Aβ and α-syn might directly interact under pathological conditions leading to the formation of toxic oligomers and nanopores that increase intracellular calcium. The interactions between Aβ and α-syn might also result in oxidative stress, lysosomal leakage, and mitochondrial dysfunction. Thus, better understanding the steps involved in the process of Aβ and α-syn aggregation is important in order to develop intervention strategies that might prevent or reverse the accumulation of toxic proteins in AD.

Interferon regulatory factor 4 (IRF4) is a transcription factor that regulates normal and malignant immune cell development and is implicated in multiple myeloma pathogenesis. This protocol describes the use of combined cell surface and intranuclear staining with fluorescent antibodies to measure IRF4 protein expression within myeloma and normal immune cells. IRF4 protein quantification may provide a valuable prognostic tool to predict disease severity and sensitivity to IRF4-targeted therapies. This flow-cytometry-based procedure could also be rapidly translated into a clinically compatible assay. For complete details on the use and execution of this protocol, please refer to Mondala et al. (2021).

Abstract Background Recent studies suggest that the pathogenic process in neurodegenerative disorders may disrupt mature neuronal circuitries and neurogenesis in the adult brain. Abnormal activation of CDK5 is associated with neurodegenerative disorders, and recently a critical role for CDK5 in adult neurogenesis has been identified. We have developed an in vitro model of abnormal CDK5 activation during adult hippocampal neurogenesis, and here we used this model to investigate aberrantly phosphorylated downstream targets of CDK5. Results Abnormal CDK5 activation in an in vitro model of adult neurogenesis results in hyperphosphorylation of collapsin-response mediator protein-2 (CRMP2) and impaired neurite outgrowth. Inhibition of CDK5, or expression of a non-phosphorylatable (S522A) CRMP2 construct reduced CRMP2 hyperphosphorylation, and reversed neurite outgrowth deficits. CRMP2 plays a role in microtubule dynamics; therefore we examined the integrity of microtubules in this model using biochemical and electron microscopy techniques. We found that microtubule organization was disrupted under conditions of CDK5 activation. Finally, to study the relevance of these findings to neurogenesis in neurodegenerative conditions associated with HIV infection, we performed immunochemical analyses of the brains of patients with HIV and transgenic mice expressing HIV-gp120 protein. CDK5-mediated CRMP2 phosphorylation was significantly increased in the hippocampus of patients with HIV encephalitis and in gp120 transgenic mice, and this effect was rescued by genetic down-modulation of CDK5 in the mouse model. Conclusions These results reveal a functional mechanism involving microtubule destabilization through which abnormal CDK5 activation and CRMP2 hyperphosphorylation might contribute to defective neurogenesis in neurodegenerative disorders such as HIV encephalitis.

Background

Deregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies.

Methods

To facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells.

Results

In lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression.

Conclusions

RNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.

In multiple myeloma, inflammatory and anti-viral pathways promote disease progression and cancer stem cell generation. Using diverse pre-clinical models, we investigated the role of interferon regulatory factor 4 (IRF4) in myeloma progenitor regeneration. In a patient-derived xenograft model that recapitulates IRF4 pathway activation in human myeloma, we test the effects of IRF4 antisense oligonucleotides (ASOs) and identify a lead agent for clinical development (ION251). IRF4 overexpression expands myeloma progenitors, while IRF4 ASOs impair myeloma cell survival and reduce IRF4 and c-MYC expression. IRF4 ASO monotherapy impedes tumor formation and myeloma dissemination in xenograft models, improving animal survival. Moreover, IRF4 ASOs eradicate myeloma progenitors and malignant plasma cells while sparing normal human hematopoietic stem cell development. Mechanistically, IRF4 inhibition disrupts cell cycle progression, downregulates stem cell and cell adhesion transcript expression, and promotes sensitivity to myeloma drugs. These findings will enable rapid clinical development of selective IRF4 inhibitors to prevent myeloma progenitor-driven relapse.

Abstract: Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent. Disclosures: Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.