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"Rodriguez, Diego J."
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Systematic profiling of conditional pathway activation identifies context-dependent synthetic lethalities
The paradigm of cancer-targeted therapies has focused largely on inhibition of critical pathways in cancer. Conversely, conditional activation of signaling pathways as a new source of selective cancer vulnerabilities has not been deeply characterized. In this study, we sought to systematically identify context-specific gene-activation-induced lethalities in cancer. To this end, we developed a method for gain-of-function genetic perturbations simultaneously across ~500 barcoded cancer cell lines. Using this approach, we queried the pan-cancer vulnerability landscape upon activating ten key pathway nodes, revealing selective activation dependencies of MAPK and PI3K pathways associated with specific biomarkers. Notably, we discovered new pathway hyperactivation dependencies in subsets of
APC
-mutant colorectal cancers where further activation of the WNT pathway by
APC
knockdown or direct β-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. Together, this study reveals a new class of conditional gene-activation dependencies in cancer.
Gain-of-function perturbation screens across 488 barcoded cell lines identify context-specific activation lethalities. The authors show that cells with MAPK, PI3K and WNT pathway activation are vulnerable to mutations that lead to further activation, suggesting a new strategy for treating tumors driven by these oncogenic pathways.
Journal Article
Comparative optimization of combinatorial CRISPR screens
Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual
Streptococcus pyogenes
Cas9 (spCas9), orthogonal spCas9 and
Staphylococcus aureus
(saCas9), and enhanced Cas12a from
Acidaminococcus
were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.
Combinatorial CRISPR screens can be utilized to identify genetic interactions and functional redundancies of multiple genes. Here, the authors benchmark ten digenic CRISPR technologies and identify novel Cas9 tracrRNA combinations that show superior performance.
Journal Article
Identification of potent biparatopic antibodies targeting FGFR2 fusion–driven cholangiocarcinoma
Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and predict response to FGFR kinase inhibitors. However, response rates and durability are limited due to the emergence of resistance, typically involving FGFR2 kinase domain mutations, and to suboptimal dosing, relating to drug adverse effects. Here, we develop biparatopic antibodies targeting the FGFR2 extracellular domain (ECD) as candidate therapeutics. Biparatopic antibodies can overcome drawbacks of bivalent monospecific antibodies, which often show poor inhibitory or even agonist activity against oncogenic receptors. We show that oncogenic transformation by FGFR2 fusions requires an intact ECD. Moreover, by systematically generating biparatopic antibodies targeting distinct epitope pairs in FGFR2 ECD, we identified antibodies that effectively block signaling and malignant growth driven by FGFR2 fusions. Importantly, these antibodies demonstrate efficacy in vivo, synergy with FGFR inhibitors, and activity against FGFR2 fusions harboring kinase domain mutations. Thus, we believe that biparatopic antibodies may serve as an innovative treatment option for patients with FGFR2-altered cholangiocarcinoma.
Journal Article
Water-related infrastructure investments in a changing environment: a perspective from the World Bank
At present, there is a global deficit in infrastructure and the World Bank Group (WBG) is one of the major sources of financing to reduce this gap worldwide. The WBG has policies and protocols for approving investments taking into consideration financial and economic indicators while ensuring social and environmental safeguards. In recent years, these safeguards have been updated to include the effects of climate change and robustness and resilience to support climate-informed project investment decision-making. A series of tools for screening projects for climate vulnerabilities and identification of risk management options have been developed to help project teams comply with these requirements. One of these tools is the hierarchical four-phased Decision Tree Framework (DTF) that, beyond screening, helps to analyze plans and project vulnerabilities, climate-related or otherwise, using a decision scaling approach, and explore risk management options, if necessary. The four phases of the DTF are (i) project screening, (ii) initial analysis, (iii) stress test, and (iv) climate risk management. This paper reviews applications of the DTF from the climate change screening phase to non-climate uncertainty screening and decision-making for project investments and prioritization. A peek into work in progress for incorporating resilience in the decision-making process, both for projects and through projects, is also provided, as well as next steps, looking forward.
Journal Article
Pharmacological restoration of GTP hydrolysis by mutant RAS
Approximately 3.4 million patients worldwide are diagnosed each year with cancers that have pathogenic mutations in one of three RAS proto-oncogenes (
KRAS, NRAS
and
HRAS
)
1
,
2
. These mutations impair the GTPase activity of RAS, leading to activation of downstream signalling and proliferation
3
,
4
,
5
–
6
. Long-standing efforts to restore the hydrolase activity of RAS mutants have been unsuccessful, extinguishing any consideration towards a viable therapeutic strategy
7
. Here we show that tri-complex inhibitors—that is, molecular glues with the ability to recruit cyclophilin A (CYPA) to the active state of RAS—have a dual mechanism of action: not only do they prevent activated RAS from binding to its effectors, but they also stimulate GTP hydrolysis. Drug-bound CYPA complexes modulate residues in the switch II motif of RAS to coordinate the nucleophilic attack on the γ-phosphate of GTP in a mutation-specific manner. RAS mutants that were most sensitive to stimulation of GTPase activity were more susceptible to treatment than mutants in which the hydrolysis could not be enhanced, suggesting that pharmacological stimulation of hydrolysis potentiates the therapeutic effects of tri-complex inhibitors for specific RAS mutants. This study lays the foundation for developing a class of therapeutics that inhibit cancer growth by stimulating mutant GTPase activity.
Tri-complex inhibitors—molecular glues with the ability to recruit cyclophilin A to the active state of RAS—stimulate GTP hydrolysis in a mutant-specific manner.
Journal Article
Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons
Odorant receptors (OR) are strongly implicated in coalescence of olfactory sensory neuron (OSN) axons and the formation of olfactory bulb (OB) glomeruli. However, when ORs are first expressed relative to basal cell division and OSN axon extension is unknown. We developed an in vivo fate-mapping strategy that enabled us to follow OSN maturation and axon extension beginning at basal cell division. In parallel, we mapped the molecular development of OSNs beginning at basal cell division, including the onset of OR expression. Our data show that ORs are first expressed around 4 d following basal cell division, 24 h after OSN axons have reached the OB. Over the next 6+ days the OSN axons navigate the OB nerve layer and ultimately coalesce in glomeruli. These data provide a previously unidentified perspective on the role of ORs in homophilic OSN axon adhesion and lead us to propose a new model dividing axon extension into two phases. Phase I is OR-independent and accounts for up to 50% of the time during which axons approach the OB and begin navigating the olfactory nerve layer. Phase II is OR-dependent and concludes as OSN axons coalesce in glomeruli.
Significance The constant generation of olfactory sensory neurons throughout life makes the system appealing for studies of the mechanisms of axon extension and connectivity. Understanding the mechanisms leading to the genesis of these new neurons is fundamental for the development of therapeutic treatments. We provide here, to our knowledge, the first detailed analysis of the sequential steps leading toward fully differentiated sensory neurons. We show that odorant receptors are not involved in the initial steps of differentiation, but only later in the final process of maturation. Moreover, the proteins that we studied here also have been implicated in normal and pathophysiological events ranging from kidney function to cancer development, making our data valuable across different disciplines.
Journal Article
Functional expression of the olfactory signaling system in the kidney
Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (Golf), are expressed in the kidney. AC3 and Golf colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3⁻/⁻ mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.
Journal Article
Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers
Although single-gene perturbation screens have revealed a number of new targets, vulnerabilities specific to frequently altered drivers have not been uncovered. An important question is whether the compensatory relationship between functionally redundant genes masks potential therapeutic targets in single-gene perturbation studies. To identify digenic dependencies, we developed a CRISPR paralog targeting library to investigate the viability effects of disrupting 3,284 genes, 5,065 paralog pairs and 815 paralog families. We identified that dual inactivation of
DUSP4
and
DUSP6
selectively impairs growth in
NRAS
and
BRAF
mutant cells through the hyperactivation of MAPK signaling. Furthermore, cells resistant to MAPK pathway therapeutics become cross-sensitized to
DUSP4
and
DUSP6
perturbations such that the mechanisms of resistance to the inhibitors reinforce this mechanism of vulnerability. Together, multigene perturbation technologies unveil previously unrecognized digenic vulnerabilities that may be leveraged as new therapeutic targets in cancer.
A CRISPR paralog targeting library profiling 815 paralog families across 11 cell lines identifies
DUSP4
and
DUSP6
as paralog pairs whose combined inactivation confers sensitivity to cells resistant to MAPK inhibitors or cells harboring
NRAS
or
BRAF
mutations.
Journal Article
Plasma membrane insertion of KCa2.3 (SK3) is dependent upon the SNARE proteins, syntaxin-4 and SNAP23
We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23.
Journal Article