Oncogene EVI1 drives acute myeloid leukemia via a targetable interaction with CTBP2.

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

Specific Targeting of Multiple Myeloma by Dual Split-signaling Chimeric Antigen Receptor T cells Directed against CD38 and CD138.

PURPOSE: The success of B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells illustrates the potential of this novel therapy for multiple myeloma. Nonetheless, broadening CAR T-cell therapy beyond BCMA requires inventive strategies as there are only a few multiple myeloma- or plasma cell-specific target antigens. We investigated the feasibility of achieving multiple myeloma specificity by dual-split CD38/CD138 CAR targeting, whereby the stimulatory and costimulatory signals for T-cell activation are split into two separate stimulatory (sCAR) and costimulatory CARs (cCAR). EXPERIMENTAL DESIGN: Using various combinations of CD38 and CD138 sCARs and cCARs with different affinities, we generated several dual-split CAR T cells and analyzed them for multiple myeloma-specific effector functions in vitro. The best-functioning CAR T cells were tested in vivo in a murine xenograft model. RESULTS: We found optimal designs of both CD38sCAR/CD138cCAR and CD138sCAR/CD38cCAR combinations, that effectively lysed multiple myeloma cells but spared single CD38- or CD138-positive healthy hematopoietic cells. While the CD38sCAR/CD138cCAR T cells achieved multiple myeloma-specific activity solely due to the low affinity of the CD38sCARs, the multiple myeloma-specific cytotoxicity, cytokine release, and proliferation of CD138sCAR/CD38cCAR T cells were established through a true combinatorial stimulatory and costimulatory effect. The most optimal combination comprised a low-affinity CD138sCAR combined with a high-affinity CD38cCAR. These CD138sCAR/CD38cCAR T cells also showed dual-antigen specific anti-multiple myeloma effects in vivo. Importantly, they were also effective against multiple myeloma cells from daratumumab pretreated patients with decreased CD38 expression levels. CONCLUSIONS: We demonstrate the possibility to specifically target multiple myeloma cells, even after CD38 targeted therapy, with carefully-designed dual-split CARs directed against CD38 and CD138.

Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence.

Despite the high remission rates achieved using T cells bearing a chimeric antigen receptor (CAR) against hematogical malignancies, there is still a considerable proportion of patients who eventually experience tumor relapse. Clinical studies have established that mechanisms of treatment failure include the down-regulation of target antigen expression and the limited persistence of effective CAR T cells. We hypothesized that dual targeting mediated by a CAR and a chimeric costimulatory receptor (CCR) could simultaneously enhance T cell cytotoxicity and improve durability. Concomitant high-affinity engagement of a CD38-binding CCR enhanced the cytotoxicity of BCMA-CAR and CD19-CAR T cells by increasing their functional binding avidity. In comparison to second-generation BCMA-CAR or CD19-CAR T cells, double-targeted CAR + CD38-CCR T cells exhibited increased sensitivity to recognize and lyse tumor variants of multiple myeloma and acute lymphoblastic leukemia with low antigen density in vitro. In addition, complimentary costimulation by 4-1BB and CD28 endodomains provided by the CAR and CCR combination conferred increased cytokine secretion and expansion and improved persistence in vivo. The cumulatively improved properties of CAR + CCR T cells enabled the in vivo eradication of antigen-low tumor clones, which were otherwise resistant to treatment with conventional CAR T cells. Therefore, multiplexing targeting and costimulation through the combination of a CAR and a CCR is a powerful strategy to improve the clinical outcomes of CAR T cells by enhancing cytotoxic efficacy and persistence, thus preventing relapses of tumor clones with low target antigen density.

Bone Marrow Mesenchymal Stromal Cells Can Render Multiple Myeloma Cells Resistant to Cytotoxic Machinery of CAR T Cells through Inhibition of Apoptosis.

PURPOSE: The microenvironment of multiple myeloma (MM) can critically impair therapy outcome, including immunotherapies. In this context, we have earlier demonstrated that bone marrow mesenchymal stromal cells (BMMSC) protect MM cells against the lytic machinery of MM-reactive cytotoxic T cells (CTL) and daratumumab-redirected natural killer (NK) cells through the upregulation of antiapoptotic proteins Survivin and Mcl-1 in MM cells. Here, we investigated the significance of this mode of immune escape on T cells engineered to express chimeric antigen receptors (CAR T cells). EXPERIMENTAL DESIGN: We tested the cytolytic ability of a panel of 10 BCMA-, CD38-, and CD138-specific CAR T cells with different affinities against a model MM cell line and against patient-derived MM cells in the presence versus absence of BMMSCs. RESULTS: Although BMMSCs hardly protected MM cells from lysis by high-affinity, strongly lytic BCMA- and CD38-CAR T cells, they significantly protected against lower affinity, moderately lytic BCMA-, CD38-, and CD138-specific CAR T cells in a cell-cell contact-dependent manner. Overall, there was a remarkable inverse correlation between the protective ability of BMMSCs and the lytic activity of all CAR T cells, which was dependent on CAR affinity and type of costimulation. Furthermore, BMMSC-mediated resistance against CAR T cells was effectively modulated by FL118, an inhibitor of antiapoptotic proteins Survivin, Mcl-1, and XIAP. CONCLUSIONS: These results extend our findings on the negative impact of the microenvironment against immunotherapies and suggest that outcome of CAR T cell or conventional CTL therapies could benefit from inhibition of antiapoptotic proteins upregulated in MM cells through BMMSC interactions.

Bone Marrow Mesenchymal Stromal Cell-mediated Resistance in Multiple Myeloma Against NK Cells can be Overcome by Introduction of CD38-CAR or TRAIL-variant.

We have recently shown the strong negative impact of multiple myeloma (MM)-bone marrow mesenchymal stromal cell (BMMSC) interactions to several immunotherapeutic strategies including conventional T cells, chimeric antigen receptor (CAR) T cells, and daratumumab-redirected NK cells. This BMMSC-mediated immune resistance via the upregulation of antiapoptotic proteins in MM cells was mainly observed for moderately cytotoxic modalities. Here, we set out to assess the hypothesis that this distinct mode of immune evasion can be overcome by improving the overall efficacy of immune effector cells. Using an in vitro model, we aimed to improve the cytotoxic potential of KHYG-1 NK cells toward MM cells by the introduction of a CD38-specific CAR and a DR5-specific, optimized TRAIL-variant. Similar to what have been observed for T cells and moderately lytic CAR T cells, the cytolytic efficacy of unmodified KHYG-1 cells as well as of conventional, DR5-agonistic antibodies were strongly reduced in the presence of BMMSCs. Consistent with our earlier findings, the BMMSCs protected MM cells against KHYG-1 and DR5-agonistic antibodies by inducing resistance mechanisms that were largely abrogated by the small molecule FL118, an inhibitor of multiple antiapoptotic proteins including Survivin, Mcl-1, and XIAP. Importantly, the BMMSC-mediated immune resistance was also significantly diminished by engineering KHYG-1 cells to express the CD38-CAR or the TRAIL-variant. These results emphasize the critical effects of microenvironment-mediated immune resistance on the efficacy of immunotherapy and underscores that this mode of immune escape can be tackled by inhibition of key antiapoptotic molecules or by increasing the overall efficacy of immune killer cells.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Polymeric micelles loaded with carfilzomib increase tolerability in a humanized bone marrow-like scaffold mouse model.

Carfilzomib-loaded polymeric micelles (CFZ-PM) based on poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) were prepared with the aim to improve the maximum tolerated dose of carfilzomib in a "humanized" bone marrow-like scaffold model. For this, CFZ-PM were prepared and characterized for their size, carfilzomib loading and cytotoxicity towards multiple myeloma cells. Further, circulation and tumor & tissue distribution of fluorescently labeled micelles were determined. Tolerability of CFZ-PM versus the clinical approved formulation - Kyprolis® - was assessed. CFZ-PM presented small diameter below 55 nm and low PDI < 0.1. Cy7-labeled micelles circulated for extended periods of time with over 80% of injected dose in circulation at 24 h after intravenous injection and 1.3% of the injected dose of Cy7-labeled micelles accumulated in myeloma tumor-bearing scaffolds. Importantly, CFZ-PM were well tolerated whereas Kyprolis® showed adverse effects. Kyprolis® dosed at the maximum tolerated dose, as well as CFZ-PM, did not show therapeutic benefit, while multiple myeloma cells showed sensitivity in vitro, underlining the importance of the bone marrow crosstalk in testing novel formulations. Overall, this work indicates that PM are potential drug carriers of carfilzomib.

A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.

PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells.

PURPOSE: Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN: We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS: We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS: A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 µmol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.

Liposomal dexamethasone inhibits tumor growth in an advanced human-mouse hybrid model of multiple myeloma.

Glucocorticoids are the cornerstone in the clinic for treatment of hematological malignancies, including multiple myeloma. Nevertheless, poor pharmacokinetic properties of glucocorticoids require high and frequent dosing with the off-target adverse effects defining the maximum dose. Recently, nanomedicine formulations of glucocorticoids have been developed that improve the pharmacokinetic profile, limit adverse effects and improve solid tumor accumulation. Multiple myeloma is a hematological malignancy characterized by uncontrolled growth of plasma cells. These tumors initiate increased angiogenesis and microvessel density in the bone marrow, which might be exploited using nanomedicines, such as liposomes. Nano-sized particles can accumulate as a result of the increased vascular leakiness at the bone marrow tumor lesions. Pre-clinical screening of novel anti-myeloma therapeutics in vivo requires a suitable animal model that represents key features of the disease. In this study, we show that fluorescently labeled long circulating liposomes were found in plasma up to 24 h after injection in an advanced human-mouse hybrid model of multiple myeloma. Besides the organs involved in clearance, liposomes were also found to accumulate in tumor bearing human-bone scaffolds. The therapeutic efficacy of liposomal dexamethasone phosphate was evaluated in this model showing strong tumor growth inhibition while free drug being ineffective at an equivalent dose (4 mg/kg) regimen. The liposomal formulation slightly reduced total body weight of myeloma-bearing mice during the course of treatment, which appeared reversible when treatment was stopped. Liposomal dexamethasone could be further developed as monotherapy or could fit in with existing therapy regimens to improve therapeutic outcomes for multiple myeloma.

CD38-targeting antibodies in multiple myeloma: mechanisms of action and clinical experience.

INTRODUCTION: Multiple myeloma (MM) is generally an incurable hematological malignancy with heterogeneous overall survival rates ranging from a few months to more than 10 years. Survival is especially poor for patients who developed disease that is refractory to immunomodulatory drugs and proteasome inhibitors. Areas covered: This review will discuss the importance of CD38-targeting antibodies for the treatment of MM patients to improve their outcome. Expert commentary: Intense immuno-oncological laboratory research has resulted in the development of functionally active monoclonal antibodies against cell surface markers present on MM cells. In this respect, CD38-targeting antibodies such as daratumumab, MOR202, and isatuximab, have high single agent activity in heavily pretreated MM patients by virtue of their pleiotropic mechanisms of action including Fc-dependent effector mechanisms and immunomodulatory activities. Importantly, CD38-targeting antibodies are well tolerated, with infusion reactions as most frequent adverse event. Altogether, this makes them attractive combination partners with other anti-MM agents. Daratumumab is already approved as monotherapy and in combination with lenalidomide-dexamethasone as well as bortezomib-dexamethasone in pretreated MM patients. Furthermore, results from studies evaluating CD38-targeting antibodies in newly diagnosed MM patients are also promising, indicating that CD38-targeting antibodies will be broadly used in MM, resulting in further improvements in survival.

Monocytes and Granulocytes Reduce CD38 Expression Levels on Myeloma Cells in Patients Treated with Daratumumab.

Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction.Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed.Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab-CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138.Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance.The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498-511. ©2017 AACR.

Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models.

Recently, NOD-SCID IL2Rγ-/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties. To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). In vitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34+ cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. In vivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34+ cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis.

A Rational Strategy for Reducing On-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors by Affinity Optimization.

Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using "light-chain exchange" technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated ∼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of ∼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs.

Establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches.

To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.