Comparison of our data obtained using computational methods

Comparison of our data obtained using computational methods and findings obtained using experimental approaches, based on structural and biochemical characterisation, shows remarkable consistency (Figs. 2A and 3). In particular, the computational study (Table 1 and Supplemental Tables S4 and S7) emphasizes the importance of using ensemble MD simulations and thermodynamic integration calculations to overcome the insufficiencies of conformational sampling in single simulations, so as to generate accurate and reproducible results even in cases (such as binding of TKI258) where the free energy difference is small. Together with the enhanced sampling (Fig. 3), this methodology shows potential for wider application in studies of drug binding and in assessments of functional and mechanistic impacts of disease mutations. In turn, these approaches can improve the ability to perform molecular-based patient selection that would assist clinical trials and subsequent treatment.
Authors\' Contributions
Role of the Funding Sources
Acknowledgements The MK laboratory is supported by Cancer Research UK (A16567). MAK and SVW were supported by Yorkshire Cancer Research (L367). We also acknowledge support from the synchrotrons DIAMOND, Soleil and ESRF. We are grateful to Alex Breeze and Navratna Vajpai for their help with recording and assigning NMR spectra. The computational research was supported by the EU FP7 p-medicine (ICT-2009.5.3) project to PVC. This work made use of HECToR and ARCHER, the UK\'s national high-performance computing service, funded by the Office of Science and Technology through EPSRC\'s High-End Computing Programme. Access to HECToR and ARCHER was provided through the EPSRC 2020 Science programme (EP/I017909/1).
Introduction Hematopoietic stem cell transplantation (HSCT) has historically been successful in treating patients with cancer, autoimmune disease (multiple sclerosis), and genetic disorders (thalassemia, sickle cell disease) (Li and Sykes, 2012). Prior to HSCT, an intense conditioning regimen is essential to reduce host tumor burden and/or auto-reactive lymphocytes. The pro-inflammatory state triggered by pre-transplant conditioning also enhances T and NK cell killing of residual tumor order thip particularly in the allogeneic setting (Paulos et al., 2007). However, this same inflammatory milieu can promote donor or host T-cell reactions that culminate in Graft-versus-Host-Disease (GvHD) or in graft rejection (Shlomchik, 2007, Ferrara et al., 2009). Many patients who require HSCT do not have an appropriate human leukocyte antigen (HLA) matched donor available. Additionally, HLA mismatch can be beneficial for cancer patients as an HLA mismatch results in increased NK cell activity (Davies et al., 2002, Ruggeri et al., 2002). Therefore, a major goal for optimizing HSCT is to enhance the engraftment of HLA mismatched grafts, while preventing, or reducing the undesired side effects triggered by the graft, the intense preconditioning regimens, or both. SHIP1 and SHIP2 are two SH2-domain containing inositol 5′ phosphatases that oppose the activity of PI3K by converting Phosphatidyl-Inositol(3,4,5)trisphosphate to Phosphadityl Inositol(3,4)bisphosphate. PI3K promotes the survival, proliferation and effector functions in a broad range of mammalian cell types, via activation of PDK1, Akt and Tec family kinases (Yuan and Cantley, 2008). SHIP1 and SHIP2 have also recently been shown to promote survival signals through the recruitment and activation of enzymes including Akt and Irgm1 (Brooks et al., 2010; Tiwari et al., 2009). SHIP1 first emerged as a potential molecular target in HSCT when it was found that both acute bone marrow (BM) graft rejection and GvHD were compromised in SHIP1 deficient hosts (Wang et al., 2002). Improved allogeneic BM engraftment in SHIP1 deficient hosts results from a constellation of immune phenotypes that include compromised NK function (Wang et al., 2002; Wahle et al., 2006; Gumbleton et al., in press), decreased numbers of T-cells in mucosal tissues (Kerr et al., 2011; Park et al., 2014), and increased immunoregulatory cell numbers such as myeloid derived suppressor cells (MDSCs) (Ghansah et al., 2004; Paraiso et al., 2007), mesenchymal stem cells (MSC) (Iyer et al., 2014a, 2014b), and Treg cells (Collazo et al., 2009; Kashiwada et al., 2006; Locke et al., 2009). Parallel studies of the hematopoietic stem cell (HSC) compartment in SHIP1−/− mice revealed that HSCs are spontaneously mobilized to the peripheral blood due to a combined effect of increased levels of granulocyte colony stimulating factor (G-CSF) and matrix metallopetidase 9 (MMP-9), and a decrease in stromal-cell derived factor 1 (SDF1) (Hazen et al., 2009). The loss of SHIP1 generates two positive outcomes in the context of HSCT: mobilization of Hematopoietic Stem-Progenitor Cells (HS-PCs) to the periphery for harvesting, and a flux in the BM microenvironment that results in a suitable milieu for incoming donor cell engraftment. In aggregate, these studies suggested that recently identified SHIP1 inhibitors (Brooks et al., 2010, 2014; Fuhler et al., 2012) could eventually find utility in various aspects of both allogeneic and autologous HSCT.