Alkylator-Induced and Patient-Derived Xenograft Mouse Models of Therapy-Related Myeloid Neoplasms Model Clinical Disease and Suggest the Presence of Multiple Cell Subpopulations with Leukemia Stem Cell Activity

<div><p>Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their <i>de novo</i> counterparts. Here, we report the development of mouse models of t-AML/MDS. First, we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU), resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia, myelodysplasia, and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second, we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall, we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics.</p></div

Mice with AML and MDS show immunophenotypic changes in bone marrow cell composition compared to untreated age-matched control mice.

<p>Single-cell suspensions of bone marrow cells were prepared at the time of sacrifice and analyzed for relative expression of myeloid, lymphoid, and hematopoietic progenitor cell markers by flow cytometry. <b>(a)</b> Expression of myeloid and lymphoid markers in control (n = 6), AML (n = 9), and MDS (n = 8) mice. Mean percentages of live/Ter119 negative BM cells are shown with standard deviations. Mice with AML and MDS have increased frequency of myeloid cells (Gr-1+ and Mac-1+) and decreased frequency of B-lymphocyte cells (CD19+) compared to control mice (* p<0.05 for controls versus AML by 2-way ANOVA test; ** p<0.05 for controls versus MDS by 2-way ANOVA test). <b>(b-i)</b> Immunophenotypic analyses of bone marrow hematopoietic precursor populations in control (n = 16), AML (n = 8), and MDS (n = 8) mice are shown. Mean percentages with standard deviations of live <b>(b)</b>, live/lineage negative <b>(c, d)</b>, live/KLS positive <b>(e-f),</b> and live/KLS negative <b>(g-i)</b> BM cells are shown. <b>(e)</b> HSC were significantly decreased in MDS mice, but not AML mice, compared to controls (* p<0.05 by Mann-Whitney test). There were no other significant changes in hematopoietic precursor cell percentages, including lineage negative <b>(b)</b>, KLS negative <b>(c)</b>, KLS positive <b>(d)</b>, MPP <b>(f)</b>, MEP <b>(g)</b>, CMP <b>(h)</b>, and GMP <b>(i)</b>.</p

AML and MDS cases engraft NSG mice but do not transplant rapidly lethal disease.

<p>Single-cell suspensions of bone marrow cells from diseased mice were prepared at sacrifice and transplanted either as bulk or CD3-depleted samples into conditioned NSG mice (n = 3 or 5 transplants per primary mouse). Peripheral blood engraftment was monitored by flow cytometry using congenic CD45.1 (NSG) and CD45.2 (DBA/SWR) markers. <b>(a)</b> A summary of ENU-treated mice used in transplant experiments, including mouse ID, diagnosis, ENU dose received, and survival from the time of ENU-treatment is shown. <b>(b, c)</b> Scatter plots of peripheral blood engraftment 60 days after transplant, expressed as the percentage of CD45.2 positive cells out of total CD45 positive cells, is shown for eight AML <b>(b)</b> and nine MDS <b>(c)</b> cases. <b>(d)</b> Mean PB engraftment with standard deviations of all AML (n = 34) and MDS (n = 47) transplants at 60 days is shown compared to transplants from two untreated age-matched control mice. AML cases engrafted at a significantly higher percentage than MDS cases, and both AML and MDS mean engraftment levels were significantly lower than control (* p<0.05 by unpaired t test). <b>(e)</b> Kaplan-Meier survival analysis of transplanted mice demonstrates a long median overall survival for both AML (n = 34, 265 days) and MDS (n = 47, 275 days) transplants.</p

Treatment of mice with ENU leads to development of therapy-related myeloid neoplasms.

<p><b>(a)</b> Forty DBA/2J and ten SWR/J mice were treated with 100–300 mg/kg ENU, monitored for disease, and sacrificed when moribund. Diagnostic analysis revealed AML, MDS, and non-AML/MDS diseases in 16%, 22%, and 32% of mice respectively. The remaining 30% of mice died from ENU-toxicity or were not analyzable. <b>(b)</b> Kaplan-Meier survival analysis of ENU-treated (n = 35) mice shows a median overall survival of 162 days from the date of treatment. There were no statistically significant differences in survival by diagnosis (n = 8, n = 11, and n = 16 for AML, MDS, and non-AML/MDS diseases respectively). <b>(c)</b> Kaplan-Meier survival analysis of ENU-treated mice shows no significant difference in survival among the various ENU dose levels tested (n = 35, n = 9, n = 10, n = 16 for total, 100 mg/kg, 200 mg/kg, and 300 mg/kg respectively).</p

ENU-treated mice demonstrate morphologic changes consistent with AML or MDS.

<p>Smears from peripheral blood, bone marrow, spleen, and liver were prepared from mice at sacrifice and stained with Wright-Giemsa stain. Samples of tibia, spleen, liver, kidney, and brain were fixed in formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Representative smears (1000x) from an AML case show circulating peripheral blood blasts <b>(a)</b> and blasts infiltrating the bone marrow <b>(b)</b>, spleen <b>(c)</b>, and liver <b>(d).</b> Representative images (400x) show infiltrates of blasts in bone marrow <b>(e)</b>, spleen <b>(f)</b>, liver <b>(g)</b>, kidney <b>(h)</b>, and brain <b>(i)</b> sections from a mouse with AML. Representative smears (1000x) from the peripheral blood <b>(j)</b>, bone marrow <b>(k)</b>, and spleen <b>(l)</b> from a mouse with MDS show dysplastic hematopoiesis including abnormal neutrophil nuclear maturation, target cells, basophilic stippling, and large platelets in the peripheral blood, abnormal megakaryocyte nuclear morphology in the bone marrow, and bi-nucleated red cell precursors in the spleen. Examples are highlighted with arrows.</p

Human t-AML samples can form successful xenografts in NSG mice, but do not demonstrate a uniform LSC immunophenotype.

<p>CD3-depleted human AML cells were transplanted into conditioned NSG mice either as bulk or FACS purified CD34-, CD34+/CD38+, and CD34+/CD38- cell subpopulations. Peripheral blood engraftment was monitored by flow cytometry using CD45.1 (NSG) and hCD45 (human) markers. <b>(a)</b> A summary of patient disease-related information including cytogenetics and molecular alterations, disease status, prior disease, and prior therapy is shown. <b>(b-e)</b> Scatter plots of percentage PB engraftment, expressed as the percentage of hCD45 positive cells out of total CD45 positive cells, at day 120 or death <b>(b)</b> or at day 180 or death <b>(c-e)</b>, are shown. <b>(b)</b> CD3-depleted bulk AML samples from the seven t-AML cases were transplanted into conditioned NSG mice (n = 2–5 per primary t-AML sample). Three samples (SU108, SU158, and SU190) established hCD45+hCD33+ AML xenografts with moderate to high engraftment that were all fatal to the recipient mice by 127 days after transplantation. CD34-, CD34+/CD38+, and CD34+/CD38- cell subpopulations from engrafting samples (SU108, SU158, and SU190) were FACS purified and transplanted into conditioned NSG mice (n = 3–7 per cell subpopulation from each primary t-AML sample). <b>(c)</b> CD34+/CD38+ and CD34+/CD38-, but not CD34-, cells from SU108 established AML xenografts. Xenografts from both cell subpopulations were fatal to the recipient mice (circled). <b>(d)</b> Both CD34- and CD34+CD38- cells from SU158 established AML xenografts that were fatal to the recipient in three of seven mice from the CD34+CD38- cell subpopulation (circled). <b>(e)</b> All three cell subpopulations from SU190 were capable of forming AML xenografts, although only CD34- transplanted fatal disease (circled). <b>(f)</b> Mice with nonlethal engraftment had significantly higher BM engraftment and lower spleen engraftment than mice that died from t-AML xenografts (* p = 0.00396, ** p = 7.235 x 10⁻⁸ by Student’s t-test).</p

Mice with AML and MDS exhibit weight loss, splenomegaly, and blood count abnormalities at the time of sacrifice.

<p>Scatter plots with means and standard deviations for controls [n = 29, except for <b>(b)</b> where n = 8], AML (n = 8), and MDS (n = 11) are shown for: weight in g <b>(a)</b>; spleen length in cm <b>(b)</b>; WBC in 10³ cells/mm³ <b>(c)</b>; Hgb in g/dL <b>(d)</b>; MCV in fL <b>(e)</b>; and platelet count in 10³ platelets/mm³ <b>(f)</b>. * p<0.05 for controls versus AML by Mann-Whitney test; ** p<0.05 for controls versus MDS by Mann-Whitney test.</p

Reagents and Conditions Used for Immunohistochemistry.

<p>Reagents and Conditions Used for Immunohistochemistry.</p

Atypical expression of HGAL in isolated atypical follicles.

<p>An axillary lymph node in a 53-year old woman (case 2) shows a single atypical follicle with pleomorphic large cells overexpressing HGAL (A and B). Sections of the tonsil in a 6-year old boy stained with HGAL, show preservation of overall architecture with numerous normal reactive follicles and a gradient of HGAL staining with higher intensity in the dark zone. In the atypical follicle (indicated by arrow in panel D), HGAL staining is abnormal and shows overexpression throughout the affected follicle (C and D).</p

Summary of Immunohistologic Findings.

<p>Summary of Immunohistologic Findings.</p