combined pik3ca and fgfr inhibition with alpelisib and infigratinib in patients with pik3ca mutant solid tumors with or without fgfr alterations

PURPOSE Concurrent PIK3CA mutations and fibroblast growth factor receptor (FGFR) alterations occur in multiple cancer types, including estrogen receptor–positive breast cancer, bladder cancer, and endometrial cancer. In this first-in-human combination trial, we explored safety and preliminary efficacy of combining the PI3Kα selective inhibitor alpelisib with the FGFR1-4 selective inhibitor infigratinib. PATIENTS AND METHODS Patients with PIK3CA-mutant advanced solid tumors, with or without FGFR1-3 alterations, were enrolled in the dose escalation or one of three molecular-defined dose-expansion cohorts. The primary end point was the maximum tolerated dose. Secondary end points included safety, pharmacokinetics, and response. Archival tumor samples were sequenced to explore genomic correlates of response. RESULTS In combination, both agents were escalated to full, single-agent recommended doses (alpelisib, 300 mg per day continuously; infigratinib, 125 mg per day 3 weeks on followed by 1 week off). The toxicity profile of the combination was consistent with the established safety profile of each agent, although 71% of all patients required at least one treatment interruption or dose reduction. Molecularly selected dose expansions in breast cancer and other solid tumors harboring PIK3CA mutations, alone or in combination with FGFR alterations, identified sporadic responses, predominately in tumor types and genotypes previously defined to have sensitivity to these agents. CONCLUSION The combination of alpelisib and infigratinib can be administered at full single-agent doses, although the high rate of dose interruption or reduction suggests long-term tolerability may be challenging. In exploratory signal-seeking cohorts of patients harboring dual PIK3CA and FGFR1-3 alterations, no clear evidence of synergistic activity was observed

Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβx-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ1–38 occurs as Aβ1–42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders

Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of α-Synucleinopathy

Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by α-synuclein accumulation that includes dementia with Lewy bodies (DLB) and Parkinson's Disease (PD). Recent evidence suggests that impairment of lysosomal pathways (i.e. autophagy) involved in α-synuclein clearance might play an important role. For this reason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLB and Alzheimer's Disease (AD) and in α-synuclein transgenic mice.By immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor were elevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 and Beclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying α-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, and ultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations were observed in the brains of α-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injection of a lentiviral vector expressing Atg7 resulted in reduced accumulation of α-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations.This study supports the notion that defects in the autophagy pathway and more specifically in mTor and Atg7 are associated with neurodegeneration in DLB cases and α-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects

Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation

Pathogenic changes and compensatory responses in a model of age-related neurodegeneration

Lysosomes are part of a dynamic system that is involved in replenishing a variety of cellular components. The more than 40 neurodegenerative diseases that arise from lysosomal dysfunction evidence the importance of these organelles in maintaining cell health. Enzymatic deficiencies induce the deposition of abnormally processed materials believed to contribute to the characteristic mental retardation and brain damage. The fact that age-related diseases exhibit similar changes has prompted investigation of whether lysosomal dysfunction contributes to these neurodegenerative disorders. Post-mortem analyses have demonstrated evidence of such a contribution, since the presence of protein aggregates (e.g., neurofibrillary tangles and amyloid plaques in Alzheimer\u27s disease (AD) and huntingtin protein aggregates in Huntington\u27s Disease (HD)) has been correlated with evidence of lysosomal dysfunction. Although to a lesser degree, normal aging is associated with lysosomal disruption, thus establishing the “age” risk-factor in the two diseases. In vivo and in vitro models also report that experimentally-induced lysosomal dysfunction promotes abnormal protein processing. Moreover, protein aggregates and lysosomal dysfunction have been linked to synaptic deterioration and cognitive decline. However, there is a lack of mechanistic data to explain the numerous connections. The work presented here studied the effects of lysosomal perturbation in cultured hippocampal slices to understand why synapses are particularly vulnerable. A general lysosomal inhibitor was found to induce a distinct pathogenic cascade including, abnormal aggregation, protein fragmentation and microtubule destabilization. Subsequent changes included severe impairment of neuronal transport and corresponding declines in the expression of synaptic proteins. The application of a lysosomal modulator markedly up-regulated the levels of lysosomal enzymes, reversing the pathologic steps, thereby validating the identified cascade. Although smaller, lysosomal activation events have been found associated with neurodegenerative disorders and were speculated to represent a kind of compensatory response. In conclusion, the results described show that lysosomal dysfunction leads to the disruption of microtubule-based transport mechanisms vital for synaptic maintenance. They also indicate that lysosomal activation in AD and HD represents an internal repair system that (1) is activated in response to lysosomal disturbances and (2) can be pharmacologically enhanced as a potential avenue for therapeutic intervention to treat an array of neurodegenerative diseases.

Biosynthesis and turnover of DOPA-containing proteins by human cells

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in age-related diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease. © 2004 Elsevier Inc. All rights reserved