CD99 antibody disrupts T-cell acute lymphoblastic leukemia adhesion to meningeal cells and attenuates chemoresistance

Central nervous system (CNS) relapse is a significant cause of treatment failure among patients with acute lymphoblastic leukemia. In prior work we found that the meninges, the thin layer of tissue that covers the brain and spinal cord, harbor leukemia cells in the CNS. Importantly, direct interactions between leukemia and meningeal cells enabled leukemia chemoresistance. Herein, we show that an antibody targeting CD99, a transmembrane protein expressed on meningeal cells and many leukemia cells, disrupts adhesion between leukemia and meningeal cells and restores sensitivity of the leukemia cells to chemotherapy. This work identifies a mechanism regulating critical intercellular interactions within the CNS leukemia niche and may lead to novel therapeutic approaches for overcoming niche-mediated chemoresistance.

Cryopreserved human umbilical cord versus acellular dermal matrix patches for in utero fetal spina bifida repair in a pregnant rat model

OBJECTIVEDespite significant improvement in spinal cord function after in utero spina bifida (SB) repair compared with traditional postnatal repair, over half of the children who undergo this procedure do not benefit completely. This lack of benefit has been attributed to closure methods of the defect, with subsequent spinal cord tethering at the repair site. Hence, a regenerative patch or material with antiinflammatory and anti-scarring properties may alleviate comorbidities with improved outcomes. The authors’ primary objective was therefore to compare cryopreserved human umbilical cord (HUC) versus acellular dermal matrix (ADM) patches for regenerative repair of in utero SB lesions in an animal model.METHODSIn vivo studies were conducted in retinoic acid–induced SB defects in fetuses of Sprague-Dawley rats. HUC or ADM patches were sutured over the SB defects at a gestational age of 20 days. Repaired SB defect tissues were harvested after 48–52 hours. Tissue sections were immunofluorescently stained for the presence of neutrophils, macrophages, keratinocytes, meningeal cells, and astrocytes and for any associated apoptosis. In vitro meningeal or keratinocyte cell coculture experiments with the ADM and HUC patches were performed. All experiments were scored quantitatively in a blinded manner.RESULTSNeutrophil counts and apoptotic cells were lower in the HUC-based repair group (n = 8) than in the ADM patch repair group (n = 7). In the HUC patch repair group, keratinocytes were present on the outer surface of the patch, meningeal cells were present on the inner surface of the patch adjacent to the neural placode, and astrocytes were noted to be absent. In the ADM patch repair group, all 3 cell types were present on both surfaces of the patch. In vitro studies showed that human meningeal cells grew preferentially on the mesenchymal side of the HUC patch, whereas keratinocytes showed tropism for the epithelial side, suggesting an inherent HUC-based cell polarity. In contrast, the ADM patch studies showed no polarity and decreased cellular infiltration.CONCLUSIONSThe HUC patch demonstrated reduced acute inflammation and apoptosis together with superior organization in regenerative cellular growth when compared with the ADM patch, and is therefore likely the better patch material for in utero SB defect repair. These properties may make the HUC biomaterial useful as a “meningeal patch” during spinal cord surgeries, thereby potentially reducing tethering and improving on spinal cord function.

The meninges enhance leukemia survival in cerebral spinal fluid

Central nervous system (CNS) relapse is a common cause of treatment failure in patients with acute lymphoblastic leukemia (ALL) despite current CNS-directed therapies that are also associated with significant short and long-term toxicities. Herein, we showed that leukemia cells exhibit decreased proliferation, elevated reactive oxygen species (ROS), and increased cell death in CSF both in vitro and in vivo. However, interactions between leukemia and meningeal cells mitigated these adverse effects. This work expands our understanding of the pathophysiology of CNS leukemia and suggests novel therapeutic approaches for more effectively targeting leukemia cells in the CNS.

Influence of the CNS Niche on Acute Lymphoblastic Leukemia Biology

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While significant progress has been made in the therapy of leukemia, several obstacles still hinder cure. Central nervous system (CNS) relapse is a major cause of treatment failure among patients with ALL and current CNS-directed therapies are also associated with significant morbidities including neurocognitive deficits, endocrinopathies, and secondary malignancies. Consequently, novel CNS-directed leukemia therapies are needed to improve long-term outcomes in ALL while decreasing treatment-related morbidity. We developed in vitro and in vivo models of CNS leukemia in order to identify the unique characteristics of the CNS microenvironment that create a sanctuary site for leukemia cells. We transplanted multiple human ALL cell lines into immune-compromised mice (NSG). Mice were not irradiated or conditioned prior to transplantation to avoid perturbing leukemia niches. Using this xenotransplant model, we then identified the meninges as the predominant CNS site that harbors leukemia cells both before and after treatment with systemic cytarabine. This anatomic distribution of CNS leukemia agrees with other murine studies and also recapitulates the histopathology of human CNS leukemia. Having demonstrated that the meninges provide a unique niche for leukemia, we then developed ex vivo co-culture approaches to focus more specifically on the effects of the meninges on leukemia biology. To more accurately model the leukemia-meningeal niche in co-culture we substituted tissue culture media for cerebral spinal fluid (CSF). However, given the unique composition and concentration of many substrates in CSF relative to serum or media we first characterized the effects of CSF on leukemia cells. We found that leukemia cells in CSF have limited survival even when replenished daily with fresh CSF. We hypothesized this decrease in viability may be secondary to elevated reactive oxygen species (ROS) given the lower levels of redox proteins in CSF. Accordingly, we found leukemia cells in CSF showed a higher level of ROS compared to the leukemia cells in regular media. Addition of N-acetyl-cysteine, a ROS scavenger, to CSF decreased ROS levels and cell death in leukemia cells. Moreover, leukemia cells co-cultured with meningeal cells in CSF showed decreased ROS levels compared to leukemia cells growing in suspension in CSF. We found the effect of the meninges on leukemia biology extends beyond ROS regulation. Leukemia cells co-cultured with meningeal cells were also significantly more resistant to chemotherapy-induced apoptosis through effects on apoptosis balance and enhanced quiescence. These effects were reversed when the leukemia cells were removed from the meninges and placed back into suspension. Moreover, leukemia cells cultured in meningeal conditioned media also exhibited mild chemoresistance, indicating a role for a soluble factor(s) secreted by meningeal cells. Using proteomic approaches we then identified candidate soluble factor(s) secreted by meningeal cells that may contribute to leukemia chemoresistance. These results show that meningeal cells influence key aspects of leukemia biology, including ROS regulation and chemoresistance, and that the pathophysiology of CNS leukemia is not only related to the ability of leukemia cells and chemotherapy to access the restricted CNS environment. Finally we are leveraging this knowledge of the CNS leukemia niche into the development of novel CNS-directed therapies that modulate ROS levels, target candidate soluble factors, or that disrupt the interactions between leukemia and meningeal cells. Disclosures No relevant conflicts of interest to declare.

Overcoming Acute Lymphoblastic Leukemia Chemoresistance Induced By the Meninges

Central nervous system (CNS) relapse is a leading cause of treatment failure in acute lymphoblastic leukemia (ALL). We have shown that the meninges provide a unique leukemia niche in the CNS that enhances leukemia chemoresistance through effects on apoptosis and quiescence. We now describe our work to leverage this new knowledge of the CNS leukemia niche into novel CNS-directed leukemia therapies that 1) target vulnerabilities unique to leukemia cells in the meninges, 2) inhibit the meningeal pathways that contribute to leukemia chemoresistance, and 3) disrupt the interactions between leukemia and meningeal cells. First, we focused on identifying and targeting vulnerabilities unique to leukemia cells in the meninges. We used a leukemia-meningeal co-culture system and tested the ability of drugs targeting pathways important for leukemia biology to overcome meningeal-mediated leukemia resistance to cytarabine and methotrexate. Intriguingly, we found that pre-treatment of leukemia cells with ruxolitinib, a JAK1/2 inhibitor, prior to co-culture with primary meningeal cells diminished leukemia chemoresistance. Ruxolitinib had no effect on the chemosensitivity of leukemia cells in suspension. Other JAK1/2 inhibitors, such as CHZ868, yielded comparable results and we are currently testing the ability of JAK2 knockdown by shRNA in leukemia cells to overcome chemoresistance. As a potential explanation for the mechanism by which ruxolitinib overcomes meningeal-mediated leukemia resistance, we found that ruxolitinib decreases leukemia quiescence and enhances proliferation in co-culture, as measured by FxCycle and Ki67 staining. Finally, preliminary experiments in xenotransplanted mice showed that ruxolitinib significantly enhanced the efficacy of cytarabine in treating leukemia in the meninges. Second, we also identified and therapeutically targeted meningeal signaling pathways that contribute to leukemia chemoresistance. We used reverse phase protein arrays (RPPA; MD Anderson) to assess the effects of co-culture on protein expression and phosphorylation in primary meningeal cells. RPPA determines the levels of >300 proteins involved in multiple signaling pathways. Amongst other findings, we identified activation of the AKT pathway in primary meningeal cells in the presence of leukemia cells. Furthermore, pre-treatment of primary meningeal cells with INK128, an inhibitor that acts downstream of AKT and targets mTOR, prior to co-culture with leukemia cells diminished leukemia chemoresistance. Third, we tested whether disrupting meningeal-leukemia adhesion overcomes leukemia chemoresistance. Leukemia cells were dissociated from meningeal cells with trypsin and manual pipetting after co-culture for 48-72 hours. Leukemia cells were then purified with magnetic beads and placed back in suspension prior to further characterization and drug testing. We found leukemia cells placed back into suspension after co-culture with meningeal cells, or isolated from the meninges of mice, reverted back to baseline cell cycle, quiescence, and apoptosis balance characteristics. Moreover, leukemia cells removed from co-culture exhibited similar sensitivity to methotrexate and cytarabine as leukemia cells in suspension. These results suggest that drugs or biologic agents that disrupt adhesion between leukemia and meningeal cells may restore leukemia chemosensitivity in the CNS niche. Accordingly, we next identified several cell adhesion inhibitors that effectively disrupted leukemia-meningeal adhesion in co-culture. The more promising agents are currently being characterized more extensively in co-culture and tested in combination with chemotherapy in xenotransplanted mice. In summary, in order to address the need for more efficacious and less toxic therapies for CNS leukemia, we have identified mechanisms by which the meninges enhance leukemia chemoresistance. We are now testing novel strategies for overcoming leukemia chemoresistance that by targeting different components of the CNS leukemia niche (leukemia cells, meningeal cells, or the interactions between the two cell types) may be complementary or even synergistic. Disclosures No relevant conflicts of interest to declare.

Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus

ABSTRACTJC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virusin vivoare not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cellsin vitro. Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML.IMPORTANCEJCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood-cerebrospinal fluid barrier, the choroid plexus and leptomeninges are also poised to play roles in virus invasion of brain parenchyma, where infection of macroglial cells leads to the development of progressive multifocal leukoencephalopathy, a severely debilitating and often fatal infection. In this paper we show for the first time that primary choroid plexus epithelial cells and meningeal cells are infected by JCPyV, lending support to the association of JCPyV with meningoencephalopathies. These data also suggest that JCPyV could use these cells as reservoirs for the subsequent invasion of brain parenchyma.