Sajad Khazal

Effects of the small-molecule inhibitor of integrin α4, TBC3486, on pre-B-ALL cells

The treatment of patients with chemotherapy-resistant leukemia remains a challenge. A role of the microenvironment for drug resistance of leukemia cells has been proposed.1 We have identified the adhesion molecule integrin α4 as a central mediator of drug resistance of pre-B-cell acute lymphoblastic leukemia (ALL).2 We thus demonstrated that chemotherapy-resistant pre-B-ALL cells can be eradicated in a xenograft model by concurrent blockade of α4 using natalizumab, a humanized anti-α4 antibody in clinical use against multiple sclerosis,3 and Crohns Disease.2 Here, we extended our studies to an alternative α4 inhibitor, the non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicated that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of integrin α4.4 As such, the compound has shown efficacy in integrin α4-dependent models of inflammatory and autoimmune disease4 and has shown efficacy in mice with autoimmune encephalomyelitis, a model for multiple sclerosis.5 As opposed to natalizumab, which will inhibit both members of the α4 integrin family, α4β1 and α4β7, TBC3486 is 200-fold more potent in inhibiting α4β1 than α4β7. In addition, it is completely inactive against all other integrins tested, including members of the β2, β3 as well as other members of the β1 family of integrins.4 The potential usefulness of this novel inhibitor for pre-B-ALL treatment was tested in our established in vitro and in vivo assays.2, 4 We evaluated the effect of TBC3486 on de-adhesion of patient-derived ALL cells (LAX7R) using established adhesion assays. As a control for our studies, a close structural analog was used that lacks activity toward α4β1 integrin (THI0012). After activating LAX7R cells with 1 mM Mn2+, leukemia cells were co-cultured with the murine stromal cell line OP9.6, 7 Subsequently, LAX7R cells were treated with different doses of TBC3486 (5, 10 and 25 μM) and its control, THI0012 (5, 10 and 25 μM), for 4 days. TBC3486 dose-dependently inhibited adhesion of ALL cells (Figure 1a), albeit the adhesion was not completely blocked. The dose of 25 μM was selected for subsequent studies. The concentrations of compound required for inhibition in these assays are higher than previously reported.4 This is due to the fact that TB3486 is highly protein bound in the presence of 20% serum (used in these assays), which significantly reduces the amount of free compound available to bind to the integrin receptor. Next, we determined whether TBC3486 decreases binding of three xenograft cells derived from primary pre-B-ALL cases (LAX7R, ICN3 and SFO3) to the counter-receptor of α4 integrin, human VCAM-1. Adhesion assays were performed as previously described4, 8, 9 by culturing ALL samples treated with TBC3486 (25 μM) or THI0012 (25 μM) on hVCAM-1-coated plates for 2 days. Compared with the control group, TBC3486-treated ALL cells showed significantly less adhesion to hVCAM-1 (Figures 1c, e and g); however, the adhesion was not completely blocked. CD49d (MFI) is expressed with higher intensity in LAX7R compared with the other two samples (ICN3 and SFO3) (data not shown), which may explain why TBC3486 blocked a larger percentage of LAX7R adhesion to VCAM-1. In addition to blocking cell adhesion, TBC3486 treatment also specifically targeted the expression of integrin α4, but not integrin α5 and α6 (Figure 1b). The treatment with TBC3486 did not affect cell viability in all three cases (Figures 1d, f and h) compared with the THI0012 control. Taken together, TBC3486 leads to the partial de-adhesion of pre-B-ALL cells from its counter-receptor VCAM-1 under the conditions described.

Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality

Neutrophils generated by granulocyte colony‐stimulating factor (GCSF) are functionally immature and, consequently, cannot effectively reduce infection and infection‐related mortality in cancer chemotherapy‐induced neutropenia (CCIN). Am80, a retinoic acid (RA) agonist that enhances granulocytic differentiation by selectively activating transcription factor RA receptor alpha (RARα), alternatively promotes RA‐target gene expression. We found that in normal and malignant primary human hematopoietic specimens, Am80‐GCSF combination coordinated proliferation with differentiation to develop complement receptor‐3 (CR3)‐dependent neutrophil innate immunity, through altering transcription of RA‐target genes RARβ2, C/EBPε, CD66, CD11b, and CD18. This led to generation of functional neutrophils capable of fighting infection, whereas neutralizing neutrophil innate immunity with anti‐CD18 antibody abolished neutrophil bactericidal activities induced by Am80‐GCSF. Further, Am80‐GCSF synergy was evaluated using six different dose‐schedule‐infection mouse CCIN models. The data demonstrated that during “emergency” granulopoiesis in CCIN mice undergoing transient systemic intravenous bacterial infection, Am80 effect on differentiating granulocytic precursors synergized with GCSF‐dependent myeloid expansion, resulting in large amounts of functional neutrophils that reduced infection. Importantly, extensive survival tests covering a full cycle of mouse CCIN with perpetual systemic intravenous bacterial infection proved that without causing myeloid overexpansion, Am80‐GCSF generated sufficient numbers of functional neutrophils that significantly reduced infection‐related mortality in CCIN mice. These findings reveal a differential mechanism for generating functional neutrophils to reduce CCIN‐associated infection and mortality, providing a rationale for future therapeutic approaches.

Unrelated donor hematopoietic stem cell transplantation for the treatment of non‐malignant genetic diseases: An alemtuzumab based regimen is associated with cure of clinical disease; earlier clearance of alemtuzumab may be associated with graft rejection

Hematopoietic stem cell transplantation (HSCT) with matched unrelated donors (MUD), offers potentially curative therapy for patients with non‐malignant genetic diseases. In this pilot study conducted from 2006 to 2014, we report the outcomes of 15 patients with non‐malignant genetic diseases who received a myeloablative regimen with a reduced cyclophosphamide dose, adjunctive serotherapy and MUD HSCT [intravenous alemtuzumab (52 mg/m2), busulfan (16 mg/kg), fludarabine (140mg/m2), and cyclophosphamide (105 mg/kg)]. Graft‐versus‐host‐disease (GVHD) prophylaxis consisted of tacrolimus/cyclosporine and methylprednisolone. Median (range) time to neutrophil engraftment (>500 cells/µL) and platelet engraftment (>20,000/mm3) were 15 (12–28) and 25 (17–30) days, respectively. At a median follow‐up of 2 (0.2–5.4) years, the overall survival (OS) was 93.3% (95% CI: 0.61–0.99) and disease‐free survival (DFS) was 73.3% (95% CI: 0.44–0.89). Among this small sample, earlier alemtuzumab clearance was significantly associated with graft rejection (P = 0.047), earlier PHA response (P = 0.009) and a trend toward earlier recovery of recent thymic emigrants (RTE) (P = 0.06). This regimen was associated with durable donor engraftment and relatively low rates of regimen related toxicity (RRT); future alemtuzumab pharmacokinetic studies may improve outcomes, by allowing targeted alemtuzumab clearance to reduce graft rejection and promote more rapid immune reconstitution. Am. J. Hematol. 90:1021–1026, 2015.

The PI3Kδ Inhibitor Idelalisib Inhibits Homing in an in Vitro and in Vivo Model of B ALL

The quest continues for targeted therapies to reduce the morbidity of chemotherapy and to improve the response of resistant leukemia. Adhesion of acute lymphoblastic leukemia (ALL) cells to bone marrow stromal cells triggers intracellular signals that promote cell-adhesion-mediated drug resistance (CAM-DR). Idelalisib, an U.S. Food and Drug Administration (FDA)-approved PI3Kδ-specific inhibitor has been shown to be effective in CLL in down-regulating p-Akt and prolonging survival in combination with Rituximab; herein we explore the possibility of its use in B ALL and probe the mechanism of action. Primary B ALL in contact with OP9 stromal cells showed increased p-Aktser473. Idelalisib decreased p-Akt in patient samples of ALL with diverse genetic lesions. Addition of idelalisib to vincristine inhibited proliferation when compared to vincristine monotherapy in a subset of samples tested. Idelalisib inhibited ALL migration to SDF-1α in vitro and blocked homing of ALL cells to the bone marrow in vivo. This report tests PI3Kδ inhibitors in a more diverse group of ALL than has been previously reported and is the first published report of idelalisib inhibiting homing of ALL cells to bone marrow. Our data support further pre-clinical evaluation of idelalisib for the therapy of B ALL.

Management guidelines for paediatric patients receiving chimeric antigen receptor T cell therapy

In 2017, an autologous chimeric antigen receptor (CAR) T cell therapy indicated for children and young adults with relapsed and/or refractory CD19+ acute lymphoblastic leukaemia became the first gene therapy to be approved in the USA. This innovative form of cellular immunotherapy has been associated with remarkable response rates but is also associated with unique and often severe toxicities, which can lead to rapid cardiorespiratory and/or neurological deterioration. Multidisciplinary medical vigilance and the requisite health-care infrastructure are imperative to ensuring optimal patient outcomes, especially as these therapies transition from research protocols to standard care. Herein, authors representing the Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network Hematopoietic Stem Cell Transplantation (HSCT) Subgroup and the MD Anderson Cancer Center CAR T Cell Therapy-Associated Toxicity (CARTOX) Program have collaborated to provide comprehensive consensus guidelines on the care of children receiving CAR T cell therapy.Chimeric antigen receptor (CAR) T cell therapies have impressive activity in the treatment of cancer but are associated with potentially fatal toxicities. In light of the approval of CAR T cell therapy for paediatric patients, a panel of experts from the Hematopoietic Stem Cell Transplantation (HSCT) Subgroup of the Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network, the CAR T Cell Therapy-Associated Toxicity (CARTOX) Program at The University of Texas MD Anderson Cancer Center, and several other institutions have developed consensus guidelines for the use and management of these treatments in paediatric patients, which are presented herein.

Overcoming Psychosocial and Developmental Barriers to Blood and Marrow Transplantation (BMT) in an Adolescent/Young Adult (AYA) Transgender Patient with Chronic Myelogenous Leukemia

Adolescents/young adults (AYAs) afflicted with cancer face unique barriers to potentially standard curative therapies, such as blood and marrow transplantation (BMT). Transgender AYAs face additional barriers and there is a dearth of published literature regarding their oncology-related experience. We present the case of an AYA male-to-female (MTF) transgender patient on cross-sex hormone therapy, with a history of Chronic Myelogenous Leukemia (CML) and significant psychosocial barriers, which initially served as a barrier to BMT at two different centers; we modified our standard consent and education process and was able to successfully proceed with BMT and subsequently cure her CML. Despite unique challenges, AYA and transgender patients with significant psychosocial barriers may achieve successful outcomes with BMT. Research is needed regarding guidelines for cross-sex hormone therapy administration for patients undergoing BMT and other issues, which may be unique to the transgender experience.