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

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 Inca1 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, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1−/− bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1−/− 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

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

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

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

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

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

Inhibitor of CDK interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(−/−) mice. Inca1(−/−) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division

Allogeneic Transplantation Versus Chemotherapy as Postremission Therapy for Acute Myeloid Leukemia: A Prospective Matched Pairs Analysis

Purpose The majority of patients with acute myeloid leukemia (AML) who achieve complete remission (CR) relapse with conventional postremission chemotherapy. Allogeneic stem-cell transplantation (alloSCT) might improve survival at the expense of increased toxicity. It remains unknown for which patients alloSCT is preferable. Patients and Methods We compared the outcome of 185 matched pairs of a large multicenter clinical trial (AMLCG99). Patients younger than 60 years who underwent alloSCT in first remission (CR1) were matched to patients who received conventional postremission therapy. The main matching criteria were AML type, cytogenetic risk group, patient age, and time in first CR. Results In the overall pairwise compared AML population, the projected 7-year overall survival (OS) rate was 58% for the alloSCT and 46% for the conventional postremission treatment group (P = .037; log-rank test). Relapse-free survival (RFS) was 52% in the alloSCT group compared with 33% in the control group (P < .001). OS was significantly better for alloSCT in patient subgroups with nonfavorable chromosomal aberrations, patients older than 45 years, and patients with secondary AML or high-risk myelodysplastic syndrome. For the entire patient cohort, postremission therapy was an independent factor for OS (hazard ratio, 0.66; 95% CI, 0.49 to 0.89 for alloSCT v conventional chemotherapy), among age, cytogenetics, and bone marrow blasts after the first induction cycle. Conclusion AlloSCT is the most potent postremission therapy for AML and is particularly active for patients 45 to 59 years of age and/or those with high-risk cytogenetics