Kyuho Han

Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade

Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.

Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.

The Imidazopyridine Derivative JK184 Reveals Dual Roles for Microtubules in Hedgehog Signaling

Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies

Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.A CRISPR-based functional screen for Hedgehog-pathway factors identifies genes required for ciliary signaling and can be used to classify genetic disorders as ciliopathies.

E2A-PBX1 Remodels Oncogenic Signaling Networks in B-cell Precursor Acute Lymphoid Leukemia

There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR+) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR+/E2A-PBX1+ leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR.

A comprehensive portrait of cilia and ciliopathies from a CRISPR-based screen for Hedgehog signaling

The primary cilium organizes Hedgehog signaling, shapes embryonic development and is the unifying cause of the ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen robustly identifies factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovers novel components of several ciliary structures including a protein complex containing ε- and δ- tubulin that is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and reveals that many forms of congenital heart defects are ciliopathies. Collectively, this screen enables a systematic analysis of ciliary function and of ciliopathies and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

Significance Eg5, a mitotic kinesin, has long been an anticancer drug target. However, a different kinesin motor, KIF15, can rescue cell division when Eg5 is incapacitated, leading to chemotherapeutic resistance. We characterized KIF15 motor mechanics at the single-molecule level and studied the effects of combinations of small-molecule inhibitors of KIF15 and Eg5 on admixtures of motors in a motility assay, as well as on cancer cell proliferation. Taken together, our results point the way toward a strategy of combination drug therapy targeting both Eg5 and KIF15 as a means of overcoming KIF15-mediated cancer resistance. This work highlights the importance of understanding the molecular physiology of different kinesins and of exploring inhibitors that target additional mitotic kinesins. Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

SPOT-007 Identifying novel combinatorial synthetic lethal vulnerabilities in kras-driven lung cancer

Introduction KRAS is one of the most frequently mutated genes in human cancer, but the efforts to target KRAS directly have thus far been unsuccessful, highlighting the need for alternative approaches. One promising strategy is to target KRAS through synthetic lethality. However, KRAS activates multiple effector pathways, suggesting that targeting one gene may not be sufficient to fully inhibit KRAS-driven oncogenesis. Therefore, targeting combinations of genes that together are synthetic lethal with KRAS may constitute a better therapeutic approach. Material and methods To discover novel combinatorial KRAS synthetic lethal genes, we used affinity purification/mass spectrometry (AP/MS), to systematically identify KRAS interacting proteins and construct a detailed map of protein-protein interactions centred on KRAS. Based on this network we designed a CRISPR library targeting pairwise combinations of KRAS-interacting genes. Using this library we simultaneously knocked-out pairs of 119 genes in two KRAS-driven non-small cell lung cancer (NSCLC) cell lines expressing Cas9. Knock-out of several gene pairs synergistically impaired growth of these cells, while the knock-out of each of the genes alone had no or little effect. We chose 20 most promising targets for further screening in vitro and in vivo in a panel of 5 KRAS mutant and 4 wild type NSCLC cell lines. We also selected six gene pairs that had the most synergistic effect on growth for individual validation in Cas9-expressing NSCLC cells and normal human bronchial epithelial cells (HBECs). The cells were cultured in 3D, which was shown to more faithfully recapitulate important aspects of cancer biology than cells grown as monolayers. Results and discussions Out of the six gene combinations, the simultaneous knock-out of one pair of genes, Rap1GDS1 and RhoA, selectively impaired sphere growth of KRAS-dependent lung cancer cells but had little effect on the growth of KRAS-independent cells or HBECs. Moreover the individual knock-out of these genes had no effect on 3D growth in any of the cell lines, suggesting that only the combination of these two genes is synthetically lethal with KRAS. We are now performing further validation in vivo. Finally, human relevance will be determined using patient-derived xenograft (PDX) models. Conclusion Combinatorial inhibition of Rap1GDS1 and RhoA appears to be synthetically lethal with mutated KRAS and therefore these genes may constitute attractive drug targets for the treatment of KRAS-dependent NSCLC and other KRAS-driven cancers.