A Phase I Trial of Bevacizumab and Temsirolimus in Combination With Valproic Acid in Advanced Solid Tumors.

BACKGROUND: Preclinical models suggest synergy between anti-angiogenesis therapy, mammalian target of rapamycin (mTOR), and histone deacetylase inhibitors to promote anticancer activity. METHODS: This phase I study enrolled 47 patients between April 2012 and 2018 and determined safety, maximum tolerated dose (MTD), and dose-limiting toxicities (DLTs) when combining bevacizumab, temsirolimus, and valproic acid in patients with advanced cancer. RESULTS: Median age of enrolled patients was 56 years. Patients were heavily pretreated with a median of 4 lines of prior therapy. Forty-five patients (95.7%) experienced one or more treatment-related adverse events (TRAEs). Grade 3 TRAEs were lymphopenia (14.9%), thrombocytopenia (8.5%), and mucositis (6.4%). Grade 4 TRAEs included lymphopenia (2.1%) and CNS cerebrovascular ischemia (2.1%). Six patients developed DLTs across 10 dose levels with grade 3 infection, rash, mucositis, bowel perforation, elevated lipase, and grade 4 cerebrovascular ischemia. The MTD was dose level 9 (bevacizumab 5 mg/kg days 1 and 15 intravenously (IV) plus temsirolimus 25 mg days 1, 8, 15, and 22 IV and valproic acid 5 mg/kg on days 1-7 and 15-21 per orally (PO)). Objective response rate (ORR) was 7.9% with confirmed partial response (PRs) in 3 patients (one each in parotid gland, ovarian, and vaginal cancers). Stable disease (SD) ≥+6 months was seen in 5 patients (13.1%). Clinical benefit state (CBR: PR + SD ≥+6 months) was 21%. CONCLUSION: Combination therapy with bevacizumab, temsirolimus, and valproic acid was feasible, but there were numerous toxicities, which will require careful management for future clinical development (ClinicalTrials.gov Identifier: NCT01552434).

Protein inhibitors of activated STAT (Pias1 and Piasy) differentially regulate pituitary homeobox 2 (PITX2) transcriptional activity.

Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and ß-catenin. Lef-1, ß-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.

Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2.

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.

Epitope-tagging Math5 and Pou4f2: new tools to study retinal ganglion cell development in the mouse.

Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5(HA) and Pou4f2(HA) alleles. We demonstrate that the tagged alleles recapitulated the wild-type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co-staining, we found that Math5 and Pou4f2 were transiently co-expressed in newly born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope-tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development.

Ganglion cells are required for normal progenitor- cell proliferation but not cell-fate determination or patterning in the developing mouse retina.

The vertebrate retina develops from an amorphous sheet of dividing retinal progenitor cells (RPCs) through a sequential process that culminates in an exquisitely patterned neural tissue. A current model for retinal development posits that sequential cell-type differentiation is the result of changes in the intrinsic competence state of multipotent RPCs as they advance in time and that the intrinsic changes are influenced by continuous changes in the extracellular environment. Although several studies support the proposition that newly differentiated cells alter the extrinsic state of the developing retina, it is still far from clear what role they play in modifying the extracellular environment and in influencing the properties of RPCs. Here, we specifically ablate retinal ganglion cells (RGCs) as they differentiate, and we determine the impact of RGC absence on retinal development. We find that RGCs are not essential for changing the competence of RPCs, but they are necessary for maintaining sufficient numbers of RPCs by regulating cell proliferation via growth factors. Intrinsic rather than extrinsic factors are likely to play the critical roles in determining retinal cell fate.

Efficient in vivo doxycycline and cre recombinase-mediated inducible transgene activation in the murine trabecular meshwork.

PURPOSE: To generate new mouse lines that facilitate inducible gene activation in the murine trabecular meshwork in vivo. METHODS: Two expression cassettes were knocked into the 3'-UTR of the Myocilin (Myoc) locus, an abundantly expressed extracellular matrix protein produced by cells of the trabecular meshwork. The first cassette directs expression of an inducible form of Cre recombinase, CreER(T2), which is activated by tamoxifen administration under the control of endogenous Myoc regulatory elements. The second cassette contains a reverse tetracycline transactivator, rtTA(M2), which directs the expression of tetracycline-operator transgenes on exposure of animals to doxycycline (Dox). These lines were crossed to GFP and lacZ reporter mice to assay for tamoxifen or Dox-induced transgene expression. RESULTS: Both the Myoc-CreER(T2) and the Myoc-rtTA(M2) lines were capable of directing efficient and inducible expression of transgenes in the murine trabecular meshwork in vivo. In addition, activation of transgenes by Myoc-rtTA(M2) was reversible with loss of transgene expression after Dox withdrawal. Examination of multiple tissues demonstrates efficient transgene activation in the trabecular meshwork, with additional sites of transgene activation including cells in the retina, sclera, lung, kidney, and abundant activation in the neocortex and hippocampus. CONCLUSIONS: Two new mouse lines have been generated that allow for efficient and inducible transgene activation in the murine trabecular meshwork in vivo.

Overlapping spatiotemporal patterns of regulatory gene expression are required for neuronal progenitors to specify retinal ganglion cell fate.

Retinal progenitor cells (RPCs) are programmed early in development to acquire the competence for specifying the seven retinal cell types. Acquiring competence is a complex spatiotemporal process that is still only vaguely understood. Here, our objective was to more fully understand the mechanisms by which RPCs become competent for specifying a retinal ganglion cell (RGC) fate. RGCs are the first retinal cell type to differentiate and their abnormal development leads to apoptosis and optic nerve degeneration. Previous work demonstrated that the paired domain factor Pax6 and the bHLH factor Atoh7 are required for RPCs to specify RGCs. RGC commitment is marked by the expression of the Pou domain factor Pou4f2 and the Lim domain factor Isl1. We show that three RPC subpopulations can specify RGCs: Atoh7-expressing RPCs, Neurod1-expressing RPCs, and Atoh7-Neurod1-expressing RPCs. All three RPC subpopulations were highly interspersed throughout retinal development, although each subpopulation maintained a distinct temporal pattern. Most, but not all, RPCs from each subpopulation were postmitotic. Atoh7-Neurod1 double knockout mice were generated and double-mutant retinas revealed an unexpected role for Neurod1 in specifying RGC fate. We conclude that RPCs have a complex regulatory gene expression program in which they acquire competence using highly integrated mechanisms.

Canonical Wnt signaling functions in second heart field to promote right ventricular growth.

The second heart field (SHF), progenitor cells that are initially sequestered outside the heart, migrates into the heart and gives rise to endocardium, myocardium, and smooth muscle. Because of its distinct developmental history, the SHF is likely subjected to different signals from that of the first heart field. Previous experiments revealed that canonical Wnt signaling negatively regulated first heart field specification. We inactivated the obligate canonical Wnt effector beta-catenin using a beta-catenin conditional null allele and the Mef2c AHF cre driver that directs cre activity specifically in SHF. We also expressed a stabilized form of beta-catenin to model continuous Wnt signaling in SHF. Our data indicate that Wnt signaling acts in a positive fashion to promote right ventricular and interventricular myocardial expansion. Cyclin D2 and Tgfbeta2 expression was drastically reduced in beta-catenin loss-of-function mutants, indicating that Wnt signaling is required for patterning and expansion of SHF derivatives. Our findings reveal that Wnt signaling plays a major positive role in promoting growth and diversification of SHF precursors into right ventricular and interventricular myocardium.

Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development.

During vertebrate retinal development, the seven retinal cell types differentiate sequentially from a single population of retinal progenitor cells (RPCs) and organize themselves into a distinct laminar structure. The purpose of this study was to determine whether beta-catenin, which functions both as a nuclear effector for the canonical Wnt signaling pathway and as a regulator of cell adhesion, is required for retinal neurogenesis or lamination. We used the Cre-loxP system to either eliminate beta-catenin or to express a constitutively active form during retinal neurogenesis. Eliminating beta-catenin did not affect cell differentiation, but did result in the loss of the radial arrangement of RPCs and caused abnormal migration of differentiated neurons. As a result, the laminar structure was massively disrupted in beta-catenin-null retinas, although all retinal cell types still formed. In contrast to other neural tissues, eliminating beta-catenin did not significantly reduce the proliferation rate of RPCs; likewise, activating beta-catenin ectopically in RPCs did not result in overproliferation, but loss of neural retinal identity. These results indicate that beta-catenin is essential during retinal neurogenesis as a regulator of cell adhesion but not as a nuclear effector of the canonical Wnt signaling pathway. The results further imply that retinal lamination and retinal cell differentiation are genetically separable processes.

A gene network downstream of transcription factor Math5 regulates retinal progenitor cell competence and ganglion cell fate.

Math5, a mouse homolog of the Drosophila proneural bHLH transcription factor Atonal, is essential in the developing retina to establish retinal progenitor cell competence for a ganglion cell fate. Elucidating the mechanisms by which Math5 influences progenitor cell competence is crucial for understanding how specification of neuronal cell fate occurs in the retina and it requires knowledge of the downstream target genes that depend on Math5 for their expression. To date, only a handful of genes downstream of Math5 have been identified. To better define the gene network operating downstream of Math5, we used custom-designed microarrays to examine the changes in embryonic retinal gene expression caused by deletion of math5. We identified 270 Math5-dependent genes, including those that were expressed specifically either in progenitor cells or differentiated ganglion cells. The ganglion cell-specific genes included both Brn3b-dependent and Brn3b-independent genes, indicating that Math5 regulates distinct branches of the gene network responsible for retinal ganglion cell differentiation. In math5-null progenitor cells, there was an up-regulation of the proneural genes math3, neuroD, and ngn2, indicating that Math5 suppresses the production of other cell types in addition to promoting retinal ganglion cell formation. The promoter regions of many Math5-dependent genes contained binding sites for REST/NRSF, suggesting that release from general repression in retinal progenitor cells is required for ganglion cell-specific gene activation. The identification of multiple roles for Math5 provides new insights into the gene network that defines progenitor cell competence in the embryonic retina.

Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo.

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.