In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by ...irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation, by activation or transforming mutations of the p110alpha subunit, is ...associated with the development of many cancers. 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno3,2-dpyrimidine (GDC-0941) is a novel small molecule inhibitor of PI3K currently being evaluated in the clinic as an anticancer agent. The objectives of these studies were to characterize the relationships between GDC-0941 plasma concentrations and tumor reduction in MCF7.1 breast cancer xenografts and to evaluate the association between the tumor pharmacodynamic biomarker phosphorylated (p) Akt and phosphorylated proline-rich Akt substrate of 40 kDa (pPRAS40) responses and antitumor efficacy. MCF7.1 tumor-bearing mice were treated for up to 3 weeks with GDC-0941 at various doses (12.5-200 mg/kg) and dosing schedules (daily to weekly). An indirect response model fitted to tumor growth data indicated that the GDC-0941 plasma concentration required for tumor stasis was approximately 0.3 muM. The relationship between GDC-0941 plasma concentrations and inhibition of pAkt and pPRAS40 in tumor was also investigated after a single oral dose of 12.5, 50, or 150 mg/kg. An indirect response model was fitted to the inhibition of Akt and PRAS40 phosphorylation data and provided IC(50) estimates of 0.36 and 0.29 muM for pAkt and pPRAS40, respectively. The relationship between pAkt inhibition and tumor volume was further explored using an integrated pharmacokinetic biomarker tumor growth model, which showed that a pAkt inhibition of at least 30% was required to achieve stasis after GDC-0941 treatment of the MCF7.1 xenograft.
Oligonucleotide DNA microarrays were used for a genome-wide analysis of immune-challenged Drosophila infected with Gram-positive or Gram-negative bacteria, or with fungi. Aside from the expression of ...an established set of immune defense genes, a significant number of previously unseen immune-induced genes were found. Genes of particular interest include corin- and Stubble-like genes, both of which have a type II transmembrane domain; easter- and snake-like genes, which may fulfil the roles of easter and snake in the Toll pathway; and a masquerade-like gene, potentially involved in enzyme regulation. The microarray data has also helped to greatly reduce the number of target genes in large gene groups, such as the proteases, helping to direct the choices for future mutant studies. Many of the up-regulated genes fit into the current conceptual framework of host defense, whereas others, including the substantial number of genes with unknown functions, offer new avenues for research.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
BackgroundInterferon alpha 2b (IFN) is an approved immunotherapy for treatment of multiple tumors; however, the toxicity of IFNa has limited its clinical use. Using CytomX proprietary Probody® ...Therapeutics (Pb-Tx) technology, a conditionally active, mouse cross reactive IFN-a (Pb-IFNa-A/D) with minimal activity in its prodrug form, was generated to enable the study of cancer immunobiology in the mouse. Pb-IFNa-A/D is activated by the elevated protease activity associated with the tumor microenvironment (TME), leading to preferential IFN activity in the TME but not in healthy tissues. Pb-IFNa-A/D has displayed robust activity in a range of tumors, including checkpoint non-responsive models.MethodsThe Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a substrate-containing linker between the molecule and the mask by tumor-associated proteases. To investigate the pharmacodynamic activity and evaluate biomarkers related to response to Pb-IFNa-A/D, we screened 25 syngeneic murine tumor models with both monotherapy and PD-1 checkpoint blockade combination. In addition to monitoring efficacy outcomes, tumor tissue and peripheral blood were collected 48 hours post administration for pharmacodynamic response measurement.ResultsPb-IFNa-A/D monotherapy demonstrated anti-tumor activity in a range of syngeneic tumor models, including some refractory to PD-1 blockade. Combination of Pb-IFNa-A/D and PD-1 blockade demonstrated enhanced antitumor activity in comparison to PD-1 alone. We assayed peripheral blood cytokine levels 48hrs post administration of Pb-IFNa-A/D and observed a significant increase in chemokines including CXCL10, and cytokines including CCL2/3, while PD-1 blockade showed no significant increase. The combination treatment significantly increased CXCL10 in comparison to both monotherapies.To analyze changes in lymphocyte activation we performed peripheral blood immunophenotyping. Pb-IFNa-A/D and combination treatment demonstrated a significant increase in peripheral blood lymphocyte activation by CD69 and Granzyme B staining and were correlated with response.To evaluate the on-tumor changes in response to Pb-IFNa-A/D, we performed RNA-seq on tumor tissue. We observed an increase in interferon-stimulated genes with Pb-IFNa-A/D and combination treatment. In agreement with peripheral observations, Pb-IFNa-A/D and combination treatment similarly increased Granzyme-B and CXCL10 expression.ConclusionsPb-IFNa-A/D demonstrated robust anti-tumor activity in a range of syngeneic tumor models. Pharmacodynamic activity in the periphery and tumor demonstrates an IFN-stimulated immune response.These data support Pb-IFNa-A/D as a promising clinical candidate as both a single agent and in combination with checkpoint blockade, potentially expanding their benefits to patients with unresponsive tumors.
The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway ...signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP(3) production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BackgroundCytokines have been shown to elicit broad anti-tumor activity in preclinical models. These results have translated into the approval for clinical use of IFN-alpha and IL-2 before the ...checkpoint therapy era. However, to date, the clinical success of cytokines has been limited by systemic toxicity and poor exposure. CytomX Therapeutics has developed a new class of antibodies called Probody® therapeutics (Pb-Tx), designed to widen the therapeutic window by minimizing binding to targets in healthy tissue while being preferentially activated in the tumor microenvironment (TME) by tumor-associated proteases. CytomX has applied the Pb-Tx platform across multiple modalities including traditional antibodies, antibody-drug conjugates and T-cell engaging bispecifics and has advanced multiple programs into clinical studies. Here we have expanded the Pb-Tx platform with a conditionally activated cytokine version of IFN-α2b that has the potential to improve the therapeutic index of IFN-alpha therapy and allow systemic delivery.MethodsWe engineered an IFN-α2b with a dual masking strategy using a cleavable Fc domain at one end of IFN-a2b, and a cleavable affinity peptide mask at the other end. The construct was optimized to both maximize cleavability and minimize IFN-a2b toxicity. All animal experiments were reviewed and approved by CytomX's Institutional Animal Care and Use Committee (IACUC Protocol AP303).ResultsThe optimized IFN-a2b conditionally activated cytokine strongly reduced IFN-a2b activity in vitro (5,000X) in its dual-masked form. Its activity was fully restored upon protease activation. Transcriptional profiling of in vitro treated PBMC confirmed reduction of interferon-mediated activities of the masked molecule. In vitro studies with dissociated tumors indicated its ability to activate tumor immune infiltrate, that could be further enhanced by concomitant PD-L1 blockade. In mouse xenograft studies, conditionally activated IFN-a2b cytokines induced complete regression at doses as low as 0.1mpk (activity comparable to peginterferon). Surrogate conditionally activated IFN-a2b molecules were also highly potent in syngeneic mice in vivo efficacy studies. Finally, we established an in vivo safety model in hamster which has been shown to be sensitive to IFN-a-mediated toxicity in the liver and bone marrow. In hamster, we showed that conditionally activated IFN-a2b cytokines are well tolerated up to 15mpk and have reduced systemic IFN-a2b mediated toxicity as compared to the unmasked cytokine.ConclusionsTaken together these preclinical data further support the development of conditionally activated IFN-a2b with the potential to improve the therapeutic index of IFN-a therapy and to enable single agent and combination treatment in multiple clinical settings.Ethics ApprovalAll animal experiments were reviewed and approved by CytomX's Institutional Animal Care and Use Committee (IACUC Protocol AP303).
The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a
Drosophila homolog of
p53 (“
...Dmp53”). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray–induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray–induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify
Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Is all cancer therapy immunotherapy? Belvin, Marcia; Mellman, Ira
Science translational medicine,
2015-Nov-25, Volume:
7, Issue:
315
Journal Article
Peer reviewed
Researchers must renew efforts to decipher how standard chemotherapies enhance the effects of targeted immunotherapeutic agents (Müller et al., this issue).
3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone (GDC-0973) is a potent and highly selective inhibitor of mitogen-activated protein ...kinase(MAPK)/extracellular signal-regulated kinase (ERK) 1/2 (MEK1/2), a MAPK kinase that activates ERK1/2. The objectives of these studies were to characterize the disposition of GDC-0973 in preclinical species and to determine the relationship of GDC-0973 plasma concentrations to efficacy in Colo205 mouse xenograft models. The clearance (CL) of GDC-0973 was moderate in mouse (33.5 ml · min(-1) · kg(-1)), rat (37.9 ± 7.2 ml · min(-1) · kg(-1)), and monkey (29.6 ± 8.5 ml · min(-1) · kg(-1)). CL in dog was low (5.5 ± 0.3 ml · min(-1) · kg(-1)). The volume of distribution across species was large, 6-fold to 15-fold body water; half-lives ranged from 4 to 13 h. Protein binding in mouse, rat, dog, monkey, and human was high, with percentage unbound, 1 to 6%. GDC-0973-related radioactivity was rapidly and extensively distributed to tissues; however, low concentrations were observed in the brain. In rats and dogs, (14)CGDC-0973 was well absorbed (fraction absorbed, 70-80%). The majority of (14)CGDC-0973-related radioactivity was recovered in the bile of rat (74-81%) and dog (65%). The CL and volume of distribution of GDC-0973 in human, predicted by allometry, was 2.9 ml · min(-1) · kg(-1) and 9.9 l/kg, respectively. The predicted half-life was 39 h. To characterize the relationship between plasma concentration of GDC-0973 and tumor growth inhibition, pharmacokinetic-pharmacodynamic modeling was applied using an indirect response model. The KC(50) value for tumor growth inhibition in Colo205 xenografts was estimated to be 0.389 μM, and the predicted clinical efficacious dose was ∼10 mg. Taken together, these data are useful in assessing the disposition of GDC-0973, and where available, comparisons with human data were made.
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