Stanley K. Liu

The hematopoietic-specific adaptor protein Gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors

BACKGROUND The adaptor protein Gads is a Grb2-related protein originally identified on the basis of its interaction with the tyrosine-phosphorylated form of the docking protein Shc. Gads protein expression is restricted to hematopoietic tissues and cell lines. Gads contains a Src homology 2 (SH2) domain, which has previously been shown to have a similar binding specificity to that of Grb2. Gads also possesses two SH3 domains, but these have a distinct binding specificity to those of Grb2, as Gads does not bind to known Grb2 SH3 domain targets. Here, we investigated whether Gads is involved in T-cell signaling. RESULTS We found that Gads is highly expressed in T cells and that the SLP-76 adaptor protein is a major Gads-associated protein in vivo. The constitutive interaction between Gads and SLP-76 was mediated by the carboxy-terminal SH3 domain of Gads and a 20 amino-acid proline-rich region in SLP-76. Gads also coimmunoprecipitated the tyrosine-phosphorylated form of the linker for activated T cells (LAT) adaptor protein following cross-linking of the T-cell receptor; this interaction was mediated by the Gads SH2 domain. Overexpression of Gads and SLP-76 resulted in a synergistic augmentation of T-cell signaling, as measured by activation of nuclear factor of activated T cells (NFAT), and this cooperation required a functional Gads SH2 domain. CONCLUSIONS These results demonstrate that Gads plays an important role in T-cell signaling via its association with SLP-76 and LAT. Gads may promote cross-talk between the LAT and SLP-76 signaling complexes, thereby coupling membrane-proximal events to downstream signaling pathways.

A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling.

A critical event in T cell receptor (TCR)-mediated signaling is the recruitment of hematopoietic-specific adaptor proteins that collect and transmit signals downstream of the TCR. Gads, a member of the Grb2 family of SH2 and SH3 domain-containing adaptors, mediates the formation of a complex between LAT and SLP-76 that is essential for signal propagation from the TCR. Here we examine the binding specificity of the Gads and Grb2 SH3 domains using peptide arrays and find that a nonproline-based R-X-X-K motif found in SLP-76 binds to the Gads carboxy-terminal SH3 domain with high affinity (K(D) = 240 +/- 45 nM). The Grb2 C-terminal SH3 domain also binds this motif, but with a 40-fold lower affinity than Gads. Single point mutations in either the relevant R (237) or K (240) completely abrogated SLP-76 association with Gads in vivo and impaired SLP-76 function. A chimeric Grb2 protein, possessing the C-terminal SH3 domain of Gads, was able to partially substitute for Gads in signaling downstream of the T cell receptor. These results provide a molecular explanation for the specific role of Gads in T cell receptor signaling, and identify a discrete subclass of SH3 domains whose binding is dependent on a core R-X-X-K motif.

A novel poly(ADP-ribose) polymerase inhibitor, ABT-888, radiosensitizes malignant human cell lines under hypoxia

The chemo- and radioresponse of tumor cells can be determined by genetic factors (e.g., those that modify cell cycle arrest, DNA damage repair or cell death) and microenvironmental factors, such as hypoxia. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that rapidly recognizes and binds to DNA breaks to facilitate DNA strand break repair. Pre-clinical data suggest that PARP inhibitors (PARPi) may potentiate the effects of radiotherapy and chemotherapy. However, it is unclear as to whether PARPi are effective against hypoxic cells. We therefore tested the role for a novel PARPi, ABT-888, as a radiosensitizing agent under hypoxic conditions. Using human prostate (DU-145, 22RV1) and non-small cell lung (H1299) cancer cell lines, we observed that ABT-888 inhibited both recombinant PARP activity and intracellular PARP activity (86% to 92% decrease in all 3 cells lines following 2.5 microM treatment). ABT-888 was toxic to both oxic and hypoxic cells. When ABT-888 was combined with ionizing radiation (IR), clonogenic radiation survival was decreased by 40-50% under oxic conditions. Under acute hypoxia, ABT-888 radiosensitized malignant cells to a level similar to oxic radiosensitivity. To our knowledge, this is the first study to demonstrate that inhibition of PARP activity can sensitize hypoxic cancer cells and the combination of IR-PARPi has the potential to improve the therapeutic ratio of radiotherapy.

The role of Gads in hematopoietic cell signalling

Gads is a member of the family of SH2 and SH3 domain containing adaptor proteins that is expressed specifically in hematopoietic cells and functions in the coordination of tyrosine kinase mediated signal transduction. Gads plays a critical role in signalling from the T cell receptor by promoting the formation of a complex between SLP-76 and LAT. This complex couples the T cell receptor to Ras through a novel pathway involving PLC-γ1, Tec family kinases, and RasGRP. Studies with Gads-deficient mice have highlighted its importance for thymocyte proliferation during T cell maturation. Emerging evidence suggests that Gads may also play additional roles in antigen-receptor signalling and receptor tyrosine kinase mediated signalling in other hematopoietic lineages. Gads is a unique member of the Grb2 adaptor family, because its activity can be regulated by caspase cleavage. Gads nucleates multi-protein complexes that are required for tyrosine kinase-dependent signalling in immune cells and may also represent a point of modulation for these pathways through the activation of caspase-dependent signalling events.

The Adaptor Protein Gads (Grb2-Related Adaptor Downstream of Shc) Is Implicated in Coupling Hemopoietic Progenitor Kinase-1 to the Activated TCR

The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-terminal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues. Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression library screening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigated the HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 inducibly associates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated in response to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed that the carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletion of the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosine phosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3 domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.

Delta-Like Ligand 4–Notch Blockade and Tumor Radiation Response

BACKGROUND The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth. METHODS Human colorectal carcinoma LS174T cells, which express the Notch ligand delta-like ligand 4 (DLL4), and human head and neck cancer FaDu cells, which do not, were grown as subcutaneous xenografts in nude mice. The mice were treated with dibenzazepine (DBZ), a γ-secretase inhibitor that blocks all Notch signaling, or a DLL4-specific blocking monoclonal antibody, alone or in combination with ionizing radiation (n = 5-10 mice per group), and response was assessed by tumor growth delay. Microbubble contrast Doppler ultrasound was used to measure tumor blood flow. Tumor Notch activity was monitored by in vivo bioluminescence from a Notch luciferase reporter. Vessel density was assessed using Chalkley vessel counting. All statistical tests were two-sided. RESULTS In LS174T xenografts, the average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% confidence interval [CI] = 3.7 to 10.1 days, P < .001). Both Notch inhibitors suppressed tumor Notch activity within 24 hours of administration compared with vehicle (change in luciferase activity, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001). Administration of the DLL4 antibody or DBZ after ionizing radiation resulted in a supra-additive growth delay compared with vehicle (vehicle vs DLL4 antibody + ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001; vehicle vs DBZ + ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001). Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel density but reduced tumor blood flow. Combination therapy with DLL4 antibody and ionizing radiation resulted in extensive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts. CONCLUSION The combination of specific DLL4-Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunctional tumor angiogenesis and extensive tumor necrosis, independent of tumor DLL4 expression.

Cloning and characterization of mPAL, a novel Shc SH2 domain-binding protein expressed in proliferating cells.

Shc adaptor proteins play a role in linking activated cell surface receptors to the Ras signaling pathway in response to receptor mediated tyrosine kinase activation. While the function of Shc in the activation of the Ras pathway via binding to Grb2 has been well characterized, it is becoming increasingly apparent that Shc participates in additional signaling pathways through interactions with other cytoplasmic proteins. Using the yeast two-hybrid system, we have identified a unique Shc binding protein designated PAL (Protein expressed in Activated Lymphocytes) with no similarity to other known proteins. mPAL binds specifically to the Shc SH2 domain and unlike previously described Shc SH2 domain-protein interactions, the association of mPAL and Shc is phosphotyrosine-independent. Both mPAL RNA and protein expression are restricted to tissues containing actively dividing cells and proliferating cells in culture. mPAL expression is induced upon growth factor stimulation and is down-regulated upon growth inhibition. This pattern, and timing of mPAL expression and its association with the Shc adaptor molecule suggests a role for this protein in signaling pathways governing cell cycle progression.

Biomarkers for DNA DSB inhibitors and radiotherapy clinical trials

Major technical advances in radiotherapy, including IMRT and image-guided radiotherapy, have allowed for improved physical precision and increased dose delivery to the tumor, with better sparing of surrounding normal tissue. The development of inhibitors of the sensing and repair of DNA double-strand breaks (DSBs) is exciting and could be combined with precise radiotherapy targeting to improve local control following radiotherapy. However, caution must be exercised in order that DSB inhibitors are combined with radiotherapy in such a manner as to preserve the therapeutic ratio by exploiting repair deficiencies in malignant cells over that of normal cells. In this review, we discuss the rationale and current approaches to targeting DSB sensing and repair pathways in combined modality with radiotherapy. We also describe potential biomarkers that could be useful in detecting functional inhibition of DSB repair in a patient’s tissues during clinical radiotherapy trials. Finally, we examine a number of issues relating to the use of DSB-inhibiting molecular agents and radiotherapy in the context of the tumor microenvironment, effects on normal tissues and the optimal timing and duration of the agent in relation to fractionated radiotherapy.

Prostate high dose-rate brachytherapy as monotherapy for low and intermediate risk prostate cancer: Early toxicity and quality-of life results from a randomized phase II clinical trial of one fraction of 19 Gy or two fractions of 13.5 Gy

BACKGROUND AND PURPOSE Multi-fraction high dose-rate (HDR) brachytherapy as monotherapy is safe and effective for low and intermediate risk prostate cancer. Two or single fraction regimens have some radiobiological rationale. The purpose is to determine toxicity and effect on health related quality of life (HRQOL) of single fraction 19Gy or 13.5Gy×2. MATERIALS AND METHODS Eligible patients had low or intermediate risk prostate cancer, prostate volume <60cc, and no androgen deprivation use. 170 patients were randomized to receive either a single 19Gy or two fractions of 13.5Gy 1week apart. HRQOL was measured using the Expanded Prostate Index Composite (EPIC), toxicity with Common Terminology for Adverse Events (CTCAE) v4.0 and urinary symptoms with the International Prostate Symptom Score (IPSS). RESULTS Median follow-up is 20months. Grade 2 urinary toxicity occurred in 51% within the first 3months and in 31% thereafter with no significant difference between treatment arms. Ten patients (6%) developed urinary retention in the acute phase, although only 4 (2.4%) required a catheter for more than 48h. One Grade 3 acute (⩽3months) and late (>3months) urinary toxicity occurred. No more than 1% had any Grade 2 GI toxicity. The 2-fraction arm had a higher occurrence of grade 2 erectile dysfunction (29% vs. 11.5%, p=0.0249) and higher IPSS scores for the first year. Mean EPIC urinary scores at 12months decreased by 4.0 and 4.6, and sexual scores decreased by 8 and 15.9 (p=0.035) in the single and 2-fraction arms, respectively. No change occurred in the bowel or hormonal domains. CONCLUSIONS Single 19Gy and 13.5Gy×2 are both well tolerated. During the first 12months, urinary symptoms and erectile dysfunction are more common in the 2-fraction arm.

Endothelial perturbations and therapeutic strategies in normal tissue radiation damage

Most cancer patients are treated with radiotherapy, but the treatment can also damage the surrounding normal tissue. Radiotherapy side-effects diminish patients’ quality of life, yet effective biological interventions for normal tissue damage are lacking. Protecting microvascular endothelial cells from the effects of irradiation is emerging as a targeted damage-reduction strategy. We illustrate the concept of the microvasculature as a mediator of overall normal tissue radiation toxicity through cell death, vascular inflammation (hemodynamic and molecular changes) and a change in functional capacity. Endothelial cell targeted therapies that protect against such endothelial cell perturbations and the development of acute normal tissue damage are mostly under preclinical development. Since acute radiation toxicity is a common clinical problem in cutaneous, gastrointestinal and mucosal tissues, we also focus on damage in these tissues.