• Meis1 is required for the maintenance of MLL-fusion gene leukemia; HLF is a key downstream mediator of Meis1.• Meis1 and HLF restrict oxidative stress; induction of oxidative phosphorylation may be therapeutic in leukemia.Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a lowoxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment. (Blood. 2015;125(16):2544-2552 IntroductionReciprocal translocations of the 11q23 locus lead to acute leukemias of both myeloid and lymphoid lineages. These leukemias are usually resistant to conventional chemotherapies. The translocation generates an oncogenic fusion protein comprised of an amino terminus derived from MLL (now called KMT2A), the gene at 11q23, fused to a carboxy terminus derived from one of several different genes. Recent studies have revealed that these MLL-fusion proteins then join higher-order protein complexes containing various transcriptional activators and/or elongating factors such as DOT1L, PAFc, and p-TEFB. 1,2 Recruitment of these complexes to genomic loci was shown to be associated with chromatin modifications and subsequent changes in gene expression. 3 Regardless of the fusion partner involved in the translocation and of the consequential protein complex assembled, the downstream genes activated by the various MLL-fusion proteins are largely identical. Data from patientderived leukemia cells and from experimental models reveal that MLLfusion proteins upregulate the expression of posterior HOX-A genes and of the HOX cofactor MEIS1. [4][5][6] Further, retroviral overexpression of a Hox gene (with few exceptions) along with Meis1 in murine hematopoietic cells induces an aggressive leukemia in transplanted mice. 7,8 Thus, MLL-fusion protein induced upregulation of HOX and MEIS1 genes is considered central to the pathology of these leukemias.Studies to test the requirement of HOX-A and MEIS1 genes in MLL-fusion leukemias have yielded mixed results. Although shRNA knockdown of HOXA9 was shown to inhibit leukemia, mice lacking Hoxa9 protein developed leukemia induced...
Although great advances have been made in understanding the pathobiology of MLL-rearranged (MLL-r) leukemias, therapies for this leukemia have remained limited, and clinical outcomes remain bleak. To identify novel targets for immunotherapy treatments, we compiled a lineage-independent MLL-r leukemia gene signature using publicly available data sets. Data from large leukemia repositories were filtered through the In-silico Human Surfaceome, providing a list of highly predicted cell surface proteins overexpressed in MLL-r leukemias. LAMP5, a lysosomal associated membrane protein, is expressed highly and specifically in MLL-r leukemia. We found that LAMP5 is a direct target of the oncogenic MLL-fusion protein. LAMP5 depletion significantly inhibited leukemia cell growth in vitro and in vivo. Functional studies showed that LAMP-5 is a novel modulator of innateimmune pathways in MLL-r leukemias. Downregulation of LAMP5 led to inhibition of NF-κB signaling and increased activation of type-1 interferon signaling downstream of Toll-like Receptor/Interleukin 1 Receptor activation. These effects were attributable to the critical role of LAMP-5 in transferring the signal flux from Interferon Signaling Endosomes to Pro-Inflammatory Signaling Endosome. Depletion of IRF7 was able to partially rescue the cell growth inhibition upon LAMP5 downregulation. Lastly, LAMP-5 was readily detected on the surface of MLL-r leukemia cells. Targeting surface LAMP-5 using an antibody-drug conjugate leads to significant cell viability decrease specifically on MLL-r leukemias. Overall, based on the limited expression throughout human tissues, we postulate that LAMP-5 could potentially serve as an immunotherapeutic target with a wide therapeutic window to treat MLL-r leukemias.
Acute leukemias with Mixed Lineage Leukemia gene (MLL, also called KMT2A) translocations are associated with poor outcomes. These leukemias are most frequently encountered in infants and as secondary malignancies, and can be either lymphoid or myeloid. In spite of aggressive treatments, including bone marrow transplantation, infants with MLL-leukemias face a grim prognosis, with predicted survival of only 20%. Novel, effective therapies are thus urgently needed. In search for molecular targets, we observed that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5, also known as C20orf103). This observation was consistent across several gene-expression-profiling studies and occurred in both ALL and AML. Moreover, data from the TCGA study on AML showed that patients with LAMP5 expression suffered worse prognosis compared to those lacking LAMP5. We first confirmed LAMP5 expression in human leukemia cell lines by immunoblot and RT-qPCR assays. We readily detected LAMP5 mRNA and protein in MLL-fusion leukemia cell lines (MV4;11, MOLM13, THP1, RS4;11, KOPN8), while no expression of LAMP5 was found in the non-MLL-cell lines (Kasumi, K562, REH). Published ChIP-seq studies on leukemia cell lines show the MLL-fusion protein directly bound at the promotor region of LAMP5. To validate that the MLL-fusion protein activates the expression of LAMP5, we transformed human CD34+ cord blood cells with an inducible (Tet-off) retrovirus carrying the MLL-AF9 fusion cDNA. In this system, addition of Doxycycline represses expression of the MLL-AF9 oncogene. We found that LAMP5 expression directly correlated with MLL-AF9 levels, with levels of both decreasing upon addition of Doxycycline. To investigate the role of LAMP5 in MLL-fusion leukemia, we studied the effect of shRNA-mediated knockdown. By screening several hairpin sequences, we identified one construct that efficiently inhibited LAMP5 expression in MLL-fusion leukemia cells but had no effect on LAMP5-negative cells, implying specificity. All MLL-fusion leukemia cell lines tested showed growth inhibition with LAMP5 knockdown. Specifically, growth of MV4;11, THP1 and MOLM13 cells was decreased by 69%, 73% and 80% respectively compared to controls (non-targeting shRNA). When cultured in semi-solid methylcellulose media for 10 days, LAMP-5-depleted MV4;11 cells formed significantly fewer colonies than control cells (64.3 ± 25.98 and 245.3 ± 27.42 colonies per 1000 cells respectively). To investigate the role of LAMP5 in leukemia-propagation in vivo, we transplanted control and LAMP5-depleted MV4;11 cells into Busulfan-conditioned immune-deficient (NRGS) mice (2x105cells/mouse). Preliminary results from bone marrow aspirates of transplanted mice at weeks 4 post-transplant showed abundant human leukemia cells in mice receiving control cells while the mice receiving LAMP5-knockdown cells showed near-absence of human leukemia cells. Collectively, these results demonstrate that LAMP5, a novel target of MLL-fusion proteins is required for the propagation of leukemia. In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it localizes to the ER-Golgi intermediate compartment (ERGIC). In leukemia cells, using immunofluorescent confocal microscopy we detected LAMP5 in the perinuclear zones where it co-localized with ERGIC-53, a marker of the ERGIC compartment. While little is known about the functions of LAMP5 in normal pDC, studies suggest that it functions as a co-chaperone with UNC93B1, a known Toll-like Receptor (TLR) chaperone, to shuttle the TLRs to their respective locations in the plasma membrane or endosomes. In ongoing experiments, we are determining the functions of LAMP5 in leukemia, including its association with UNC93B1 and with the TLR-NFKB signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we postulate that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. Disclosures No relevant conflicts of interest to declare.
Introduction Chromosomal translocations involving the Mixed Lineage Leukemia (MLL, also called KMT2A) gene account for around 80% of the acute lymphoid leukemia (ALL) and 35%-50% of the acute myeloid leukemia (AML) diagnosed in infants, and are associated with poor outcomes. Despite improvements of conventional chemotherapies and treatments, patients with MLL-rearranged leukemia have weak response to treatment and poor prognosis. There is thus an urgent need to understand the molecular pathogenesis of these leukemias to develop more effective therapies. Gene expression studies show that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5) in both ALL and AML In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it has been shown to be required for transport of TLR9 from VAMP3+/LAMP2+/LAMP1- endolysosomal vesicles to the LAMP1+ late lysosomal compartments, modulating the signaling and production of Type I interferon to TNF production. Here, we tested the hypothesis that LAMP5 is required for MLL leukemia wherein it modulates innate immune signaling. Methods and Results In previous studies, we found LAMP5 expression being restricted to MLL-fusion leukemia cell lines. Additionally, in an inducible model, LAMP5 expression directly correlated with that of the MLL-fusion protein MLL-AF9. . Knockdown on LAMP5 in MLL-fusion leukemia cells inhibited their propagation both in vitro and in vivo whereas the non-MLL leukemias were unaffected. To determine the mechanisms by which LAMP5 promotes growth in MLL leukemias, first we studied the localization of LAMP5 in these cells. Using immunofluorescent confocal microscopy we detected LAMP5 mainly in LAMP2+/LAMP1+ compartments. Additionally, LAMP5 co-localized with MYD88, a known scaffold protein required for IL1R/TLR signaling. Accordingly, LAMP5-knockdown abrogated TLR signaling as evidenced by reduced activation of JNK, MAPK p38, IRF7 and NFKB. Conversely, over-expression of LAMP5 in the non-MLL leukemia cell line Kasumi-1 led to increased activation of JNK, p38, IRF7 and NFKB, indicating that the innate-immune signaling pathway is regulated by LAMP5 expression in leukemias.r Conclusions Collectively, these results demonstrate that LAMP5 is required for the survival of MLL leukemia and that it plays an important role in the activation of the IL1/TLR signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we propose that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. Disclosures No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.