Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

<div><p>Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of <i>Inca1</i> led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, <i>Inca1</i>-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in <i>Inca1</i>-deficient cells <i>in vivo</i>. As a consequence, leukemia induction and leukemia maintenance were severely impaired in <i>Inca1^−/−</i> bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of <i>Inca1^−/−</i> in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.</p></div

Cytotoxic stress exhausts the stem cell pool in <i>Inca1</i>-deficient bone marrow.

<p><b>A.</b> The Kaplan-Meier plot illustrates the survival of <i>Inca1^+/+</i> (n = 7) and <i>Inca1^−/−</i> (n = 13) mice after weekly administration of the myeloablative agent 5-fluorouracil (5-FU). 5-FU treatment targeted predominantly proliferating bone marrow cells and led to a significantly shortened lifespan of <i>Inca1^−/−</i> mice compared to their wild type littermates (p = 0.02, log-rank). <b>B.</b> Cell cycle analysis of total bone marrow from mice treated twice with 5-FU using BrdU/PI staining and subsequent FACS analysis. <i>Inca1^−/−</i> bone marrow cells of mice that had been exposed twice to 5-FU showed more cells in S-phase than 5-FU treated wild type mice. Shown here is a representative example of three independent experiments. <b>C.</b> Histological bone sections from 5-FU treated mice showed significant depletion of hematopoietic cells in the bone marrow of <i>Inca1^−/−</i> sternum (right-hand side) with replacement by fat cells compared to wild type control mice. In contrast, hematopoietic cell numbers were only mildly decreased in wild type mice (left panel). Lower panels show higher magnifications of the areas marked in the upper panels. <b>D.</b> The total number of nucleated bone marrow cells was significantly decreased in <i>Inca1^−/−</i> mice after two cycles of 5-FU treatment (mean ± SD, p = 0.002, t-test). All mice were 16 weeks old at the time of analysis. <b>E.</b> Bone marrow cellularity in non-challenged mice did not differ between <i>Inca1^−/−</i> and <i>Inca1^+/+</i> genotypes (mice aged 70 to 425 days; mean ± SD, p = 0.83, t-test).</p

Self-renewal of AML1-ETO9a-transduced bone marrow cells is impaired in absence of <i>Inca1</i>.

<p><b>A.</b> Schematic overview about the performed transduction and transplantation experiments. Bone marrow isolated from <i>Inca1^+/+</i> or <i>Inca1^−/−</i> mice was retrovirally transduced with AML1-ETO9a-IRES-GFP, MLL-AF9-IRES-GFP, or c-myc-IRES-BCL2-IRES-mCherry. Equal numbers of positive cells were transplanted into lethally irradiated recipients, which were then subjected to different analyses. <b>B.</b> Engraftment of AML1-ETO9a-transduced <i>Inca1^+/+</i> and <i>Inca1^−/−</i> bone marrow cells was determined by FACS analysis of GFP-positive cells in the blood of transplanted mice from 4 to 40 weeks. Engraftment was initially higher in recipients transplanted with <i>Inca1^−/−</i> bone marrow cells. The number of GFP-positive cells in <i>Inca1^−/−</i> bone marrow decreased significantly from weeks 4 and 8 until week 40 (*p = 0.015 and **p = 0.04, respectively). This analysis was restricted to mice without overt leukemia. <i>Inca1^+/+</i>: n = 14 at 4 and 8 weeks, n = 9 for 32 and 40 weeks; <i>Inca1^−/−</i>: n = 10 at 4 weeks, n = 9 at 8 and 32 weeks, n = 7 at 40 weeks. <b>C.</b> Colony assays with two subsequent replatings using AML1-ETO9a-positive <i>Inca1^+/+</i> or <i>Inca1^−/−</i> bone marrow cells, respectively, that were FACS-sorted from non-leukemic transplanted mice (n = 3 from each genotype). <i>Inca1^−/−</i> bone marrow cells transduced with AML1-ETO9a formed less colonies and replated worse than transduced <i>Inca1^+/+</i> cells (1^st plating: p = 0.01; 2^nd plating: p = 0.03; 3^rd plating: n.s.). <b>D and E.</b> For a cloning efficiency assay, 1 to 300 GFP-positive <i>Inca1^+/+</i>; AML1-ETO9a or <i>Inca1^−/−</i>; AML1-ETO9a bone marrow cells from non-leukemic transplanted mice were FACS-sorted two (<b>D</b>) or six months (<b>E</b>) and lin⁻GFP⁺ cells were seeded in semi-solid medium in a 48-well plate (n = 14 for each concentration). Two months after transplantation, <i>Inca1^+/+</i> cells had a clone forming frequency of 1/31, while the frequency was much lower in <i>Inca1^−/−</i> cells (1/164; p = 0.0001) (<b>D</b>). After six months, cloning efficiency of <i>Inca1^−/−</i> cells decreased to 1/483 (<b>E</b>).</p

Inca1 is required for AML1-ETO9a-driven leukemia initiation and maintenance <i>in vivo</i>.

<p><b>A.</b> Survival curve of recipient mice which were transplanted in two independent experiments with bone marrow cells of <i>Inca1^+/+</i> and <i>Inca1^−/−</i> mice that were retrovirally transduced with AML1-ETO9a. Left-hand side: Transplantation with 100,000 AML1-ETO9a⁺ cells (both genotypes: n = 15); right-hand side: Transplantation with 250,000 AML1-ETO9a⁺ cells (<i>Inca1^+/+</i>: n = 14, <i>Inca1^−/−</i>: n = 10). Only one <i>Inca1^−/−</i> bone marrow transplanted with 250,000 AML1-ETO9a⁺ cells led to a lethal leukemic phenotype, while about half of the <i>Inca1^+/+</i> AML1-ETO9a transplanted mice died within 300 days. <b>B.</b> Bone marrow smears (upper panel) and spleen sections (lower panel) of <i>Inca1^+/+</i> and <i>Inca1^−/−</i> AML1-ETO9a transplanted mice that died of a leukemic phenotype. Arrows in the upper panels hint at leukemic blasts, indicating acute myeloid leukemia. HE stained sections revealed morphological disruption of the splenic structure and accumulation of myeloid cells in both genotypes (lower panels). <b>C.</b> Leukemic phenotypes of recipients that received AML1-ETO9a-transduced <i>Inca1^+/+</i> and <i>Inca1^−/−</i> bone marrow cells are characterized by an infiltration of bone marrow and spleen with GFP⁺c-kit⁺ leukemic blasts in both genotypes. <b>D.</b> Survival curve of secondary recipient mice which were transplanted with bone marrow cells of leukemic mice derived from the primary transplantation shown in Fig. 5A. All mice transplanted with primary leukemic <i>Inca1^+/+</i>; AML1-ETO9a bone marrow cells died within 60 days, whereas secondary recipients of <i>Inca1^−/−</i>; AML1-ETO9a cells died with an increased latency of 150 days (<i>Inca1^+/+</i>: n = 18, <i>Inca1^−/−</i>: n = 5; p = 0.001).</p

Absence of <i>Inca1</i> accelerates MLL-AF9 driven murine leukemogenesis.

<p><b>A.</b> Survival curves of recipient mice, which were transplanted with bone marrow cells of <i>Inca1^+/+</i> or <i>Inca1^−/−</i> mice that were retrovirally transduced with MLL-AF9 as depicted in Fig. 4A (n = 11 of each genotype). Cells of both genotypes led to a fatal leukemic disease with comparable latency. <b>B.</b> Survival curves of secondary recipient mice which were transplanted with 10⁶ GFP⁺ leukemic spleen cells of leukemic mice derived from the primary transplantation shown in Fig. 5B. The secondary recipients of <i>Inca1^−/−</i>; MLL-AF9 cells (n = 15) died after a significantly longer latency than mice transplanted with <i>Inca1^+/+</i>; MLL-AF9 primary blasts (n = 15; p<0.001). Secondary transplantation of <i>p16^−/−</i>; MLL-AF9 blasts also had elongated survival compared to wild type-MLL-AF9 cells (p<0.001), but to a significant less extent (<i>Inca1^−/−</i> vs <i>p16^−/</i>−: p<0.001). <b>C.</b> Colony assays using MLL-AF9/GFP⁺c-kit⁺<i>Inca1^+/+</i> or <i>Inca1^−/−</i> spleen blast, respectively. <i>Inca1^−/−</i>; MLL-AF9 blasts formed less colonies <i>Inca1^+/+</i> blasts (p = 0.05, t-test). <b>D.</b> For a cloning efficiency assay, <i>Inca1^+/+</i>; MLL-AF9 or <i>Inca1^−/−</i>; MLL-AF9 bone marrow cells from leukemia-transplanted mice were FACS-sorted and 1 to 300 c-kit⁺GFP⁺ cells were seeded in semi-solid medium in a 48-well plate. <i>Inca1^+/+</i> cells had a clone forming frequency of 1/13, while the frequency was much lower in <i>Inca1^−/−</i> cells (1/54; p = 0.0001). Shown here are the mean results of two independent experiments.</p

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

<div><p>Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.</p></div