We present an individualized systems medicine (ISM) approach to optimize cancer drug therapies one patient at a time. ISM is based on (i) molecular profi ling and ex vivo drug sensitivity and resistance testing (DSRT) of patients' cancer cells to 187 oncology drugs, (ii) clinical implementation of therapies predicted to be effective, and (iii) studying consecutive samples from the treated patients to understand the basis of resistance. Here, application of ISM to 28 samples from patients with acute myeloid leukemia (AML) uncovered fi ve major taxonomic drug-response subtypes based on DSRT profi les, some with distinct genomic features (e.g., MLL gene fusions in subgroup IV and FLT3 -ITD mutations in subgroup V). Therapy based on DSRT resulted in several clinical responses. After progression under DSRT-guided therapies, AML cells displayed signifi cant clonal evolution and novel genomic changes potentially explaining resistance, whereas ex vivo DSRT data showed resistance to the clinically applied drugs and new vulnerabilities to previously ineffective drugs. SIGNIFICANCE:Here, we demonstrate an ISM strategy to optimize safe and effective personalized cancer therapies for individual patients as well as to understand and predict disease evolution and the next line of therapy. This approach could facilitate systematic drug repositioning of approved targeted drugs as well as help to prioritize and de-risk emerging drugs for clinical testing.

Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN.

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CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor ␣1 (GFR␣1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF 2 ⅐GFR␣1 2 complex structure at 2.35 Å resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF 2 ⅐GFR␣1 2 complex and the previously published artemin 2 ⅐GFR␣3 2 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR␣ interface between the two structures, in particular ones that buttress the key conserved Arg GFR␣ -Glu ligand -Arg GFR␣ interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR␣s, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand 2 -co-receptor 2 -RET 2 heterohexamer complexes. Consistent with this, we showed that GDNF 2 ⅐GFR␣1 2 and artemin 2 ⅐GFR␣3 2 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR␣1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg 190 -Lys 202 region in GFR␣1 domain 2. This may explain how GDNF⅐GFR␣1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET. GDNF,3 originally characterized as a growth factor promoting the survival of midbrain dopaminergic neurons (1), regulates the differentiation and development of many peripheral neurons (2) and is neuroprotective (3). GDNF is also a morphogenic factor in kidney and spermatogonia development (reviewed by Airaksinen and Saarma (2)). Some clinical trials have indicated that perfusing GDNF into the putamen may be therapeutically beneficial in Parkinson disease (4). These neuroprotective and therapeutic roles have generated wide interest in the study of the GDNF signaling system.There are three other GDNF family ligands (GFLs), neurturin (NRTN (5)), artemin (ARTN (6)), and persephin (PSPN (7)), and knock-out mice experiments have made it clear that the order of biological importance is GDNF Ͼ Ͼ NRTN Ͼ ARTN Ͼ PSPN (2). They all signal primarily through the receptor tyrosine kinase RET (8). The extracellular region of RET has four cadherin-like domains and a cysteine-rich domain. Mutations in RET can cause both gain-of-function and loss-of-function diseases. In the former category are hereditary medullary thyroid carcinoma and multiple endocrine neoplasias of types 2A and 2B (9, 10), whereas Hirschsprung disease is an example of the latter (10).GFLs are distant relatives of transforming growth factor ␤ (2). Each GFL has its own co-receptor ␣: GDNF requires GFR␣1; N...

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Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor a1 (GFRa1) and activates RET receptor tyrosine kinase. GFRa1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 Å crystal structure of GFRa1 domain 3 showing a new protein fold. It is an all-a five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRa1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRa1 or in binding RET.