Clinical Trial Details
— Status: Active, not recruiting
Administrative data
| NCT number |
NCT03918655 |
| Other study ID # |
PRT-K16055 |
| Secondary ID |
|
| Status |
Active, not recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
July 7, 2019 |
| Est. completion date |
June 2025 |
Study information
| Verified date |
November 2022 |
| Source |
Assistance Publique - Hôpitaux de Paris |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
This study is an observational study of MIF involvement in retrospectively and prospectively
included adult acute myeloid leukemia (AML). Standard care samples collected at diagnosis,
after one course of treatment, at time of remission controls, and at time of relapse will be
used.
The first objective is to determine which AMLs have pre-leukemic stem cells that overexpress
MIF. Cytogenetic and molecular (NGS) profiling will be performed at diagnosis. Blood and bone
marrow plasma, as well as bone marrow mononuclear cells will be collected and stored. The
expression of MIF and its receptor (CD74 and CXCR4) will be analysed. Their prognostic value
will be also tested.
The second objective is to test whether patients in complete remission have persistent
pre-leukemic stem cells that overexpress MIF. Blood and bone marrow plasma, bone marrow
mononuclear cells from patients in complete remission will be collected. MIF, CD74, and CXCR4
expression by hematopoietic cells at time of diagnosis and remission will be compared to
determine which patients have a persistent overexpression/secretion of MIF. In the meantime,
the persistence of initiating lesions in complete remission samples will be tested by NGS,
digital PCR, FISH, or RT-PCR methods.
The third objective is to develop a pre-clinical model to target MIF in immuno-compromised
mice (NSG mice) transplanted with primary AML cells and cells with pre-leukemic lesions. TET2
depletion leads to MIF over-expression/secretion by hematopoietic cells and improved
multi-lineage NSG-repopulation capacity. MIF inhibitors and anti-MIF antibodies will be
tested in these pre-clinical TET2-depleted models. Xenotransplantation of selected primary
AML samples and xenotransplantation of TET2 depleted hematopoietic stem cells into NSG mice
will be used.
The fourth objective is to understand how MIF is deregulated in pre-leukemic stem cells and
how the MIF-dependent crosstalk between mesenchymal stromal cells (MSCs) and pre-leukemic
stem cells or normal hematopoietic cells works.
The molecular mechanisms of MIF overexpression will be analyzed in hematopoietic stem and
progenitor cells from normal and leukemic bone marrow, with a focus on cells depleted in TET2
or DNMT3A. To study the cross-talk between hematopoietic stem and progenitor cells,
pre-leukemic stem cells, and bone marrow MSCs, co-culture experiments will be performed using
available MSC cell lines and primary MSCs from healthy donors.
Description:
The study aims at studying the involvement of MIF in adult AML.
1. DESCRIPTION OF THE ELEMENT OR ELEMENTS UNDER INVESTIGATION 1.1. Measurement of MIF
concentrations Plasma and BM supernatant from patients and controls (100 age-matched controls
for blood and BM plasma, available at Tours Hospital, ClinicalTrials.gov #NCT02789839 ) will
be analyzed by ELISA. BM supernatants will be obtained after a 2-step centrifugation
procedure: 1 mL of fresh BM from AML patients or control BM will be centrifuged at 540 g for
5 min to recover viable cells in the pellet (for cytometric and expression studies); the
supernatant will be centrifuged at 1000 g/15 min/4°C to prepare the plasma. MIF
concentrations in BM and blood plasma will be systematically compared. As MIF concentrations
may be affected by different factors, including inflammation, leukocytosis, and monocytosis,
blood cell counts and plasma C-reactive protein levels will be measured and taken into
account in the interpretation of the data.
1.2. Measurement of MIF, CD74, and CXCR4 expression Hematopoietic stem cells, progenitor
cells and blasts from AML patients will be sorted according to the expression of CD34, CD38,
CD90, CD45-RA, IL-3R antigens, taking into account the initial immunophenotype of the
disease. Hematopoietic stem cells and progenitor cells from control BM samples will be sorted
similarly. MIF, CD74 and CXCR4 RNA expression will be studied by quantitative RT-PCR in
sorted cell subpopulations. The surface expression of CXCR4 and anti-CD74 as well as
intracellular CD74 will be studied by multiparametric flow with cytometry.
1.3. Analysis of MIF, CD74, CXCR4 expression, and MIF concentrations at time of complete
remission.
The analyses performed in a) and b) will be repeated in complete remission samples in 30 to
50 selected patients. Patients will be selected according to their response to therapy
(assessment of complete remission and normal blood counts), and to the genotype of their
disease (15-25 patients with either TET2 or DNMT3A mutations at diagnosis vs 15-25 patients
without these mutations). Samples will be collected after two chemotherapy courses at time of
remission evaluation. Whenever possible, bone marrow controls of patients in long term
remission (> 1 year post diagnosis) will be collected and studied. In the meantime, NGS,
ddPCR, FISH or RT-PCR techniques will be used to assess the persistence or absence of the
initial AML lesions.
It is expected that complete material will be obtained for 80% of included patients making
the objective of 240 patients achievable in less than three years. Cell purification may be
an issue in the context of AML with aberrant immunophenotype: this will be taken into account
by the use of appropriate FACS protocols. Regarding the focus on MIF pathway, it cannot rule
out that other chemokines/cytokines, or others molecules may be involved in the communication
of AML cells and their micro-environment. Thus, aliquots of BM and blood plasma will be
cryopreserved to allow a refinement of our strategy (for example to perform cytokine arrays,
ELISA, or MS analyses which are available at Saint-Antoine Hospital).
1.4. Xenotransplantation model of primary AML in NSG mice Xenotransplantation models of
primary AML cells has been developed in a cohort of 70 AMLs in which MIF expression will be
analysed, the cytogenetic and molecular genotype, and correlate these parameters to different
engrafting phenotypes.
1.5. MIF inhibition in NSG mice transplanted with AML cells and in NSG mice transplanted with
TET2-depleted or DNMT3A-depleted cord blood HSPCs The role of MIF on leukemia development in
NSG mice xenotransplanted with primary cells will be accessed. In these experiments, primary
AML patient cells from 6 patients with TET2 mutations of our cohort that engraft will be used
to generate leukemia. When leukemic development will be seen in blood (between 8 to 10
weeks), MIF expression will be measured in the serum of mice. Mice will be randomized into
three groups with 7 mice per group: One group will be treated with a commercially available
MIF inhibitor (ISO-1) and a second group with an anti MIF antibody and one group will be
treated by the vehicle for three weeks (one injection a week). Mice will then be monitored to
detect pre-leukemic engraftment and AML development using weekly blood sampling and
anti-human CD45 staining.
A model of xenotransplantation by cord blood CD34+ cells transduced with lentivectors
expressing TET2 shRNAs have been developed. These engineered CD34 positive cells showed
increased MIF expression and secretion and demonstrate enhanced multilineage NSG engraftment.
Il will be tested whether inhibition of MIF using MIF inhibitor or anti MIF antibody leads to
a correction of the enhanced multilineage NSG engraftment by TET2-depleted cells when
compared to control cells. Similar experiments will be performed with a DNMT3A-depleted model
using DNMT3A shRNAs that are currently available in the laboratory. In addition, the effects
of MIF inhibition (inhibitor and antibody) and recombinant human MIF will be analyzed in NSG
repopulation assays of unprocessed normal cord blood CD34+ cells.
1.6. MIF inhibition in NSG mice transplanted with cells from patients in complete remission
with persistent clonal hematopoiesis (DNMT3A / TET2 mutant) Complete remission samples from
six patients with persistent clonal hematopoiesis in complete remission will be selected and
treated as in a) to test whether MIF inhibition is also able to counteract NSG repopulation
by residual pre-leukemic stem cells.
It is expected that MIF inhibition will impair AML development in mice. However, stability
and side effects of anti MIF antibody or inhibitors may be an issue in treated animals. In
this case, shRNAs will be used to knock-down MIF in AML cells before injection to NSG mice.
Another issue may be the need for co-injection of MSCs at time of xenotransplantation to
increase the relevance of the model. If needed, normal MSCs and AML MSCs will be collected,
prepared, and co-injected with AML cells.
1.7. Full characterization of the up-regulation of MIF in TET2 depleted cells How TET2 might
impact gene regulation remains still not completely understood. Recently, Cao X's team has
shown that loss of Tet2 resulted in the upregulation of several inflammatory mediators. In
this model, Tet2 recruited Hdac2 and repressed transcription of Il6 via histone
deacetylation. It has been found that TET2 binds the proximal promoter of MIF gene which is
enriched in CpG islands in Kasumi cells. Loss of TET2 results in MIF accumulation in several
leukemic models through EGR1. i) MIF promoter methylation will be analyzed by sequencing
after bisulfite treatment in normal CD34+ cells (20 samples) and cell populations from AMLs
sorted as in c)(150 samples) ii) EGR1 mRNA expression will be analyzed by qRT-PCR in normal
and AML pre-leukemic stem cells to see if there is a correlation with MIF expression as
observed in other models iii) ChIP-PCR analyses focusing on MIF promoter will be performed
for EGR1, H3K4me3, H3K27me3 and HDAC2 in control and AML patient samples. If EGR1 is not
involved in this system, MIF promoter luciferase assay will be used to identify transcription
factor sites that are essential for its up-regulation in AML samples and validate the targets
by ChIP-PCR. All these technologies are available.
1.8. bidirectional communication between MSCs and AML cells or normal HSPCs The impact of MIF
overexpression in AML cells will be tested on the physiology of normal MSCs (differentiation,
sustain of hematopoiesis, production of cytokines / chemokines, interactome). For this aim,
normal primary MSCs from adult bone marrow (already available) will be isolated and expanded
as described (Reinisch A et al, Blood 2015, 125:249-260). The Cells will be amplified up to
passage 2 (P2). MSCs will be treated with different concentrations of recombinant MIF for 24
and 48 hours and the proliferation, production of cytokines/ chemokines will be evaluated. As
it has been reported that MIF stimulates glycolysis, the effects of MIF will also decipher
will be also deciphered in the energetic metabolism of MSCs by measuring mitochondrial
respiration and glycolysis of MSCs (Seahorse XF extracellular flux analyzer) and by
determining their MIF-induced metabolic signature using high throughput multiplexed
fingerprinting (Omnilog Biolog). To model the effects of MIF overexpression by AML cells,
will perform co-culture experiments between human primary AML cells (5 samples) that express
or not (shRNA) of MIF. After 24 and 48 hours, MSCs will be sorted and their proliferation,
cell cycle and differentiation potential into osteoblasts, adipocytes and chondrocytes will
be evaluated. Previous studies indicate that MIF-CXCR4 is a novel axis for MSC recruitment to
tumors in vivo.
Thus it will be studied whether recombinant MIF may recruit MSCs using in vitro transwell
assays. Effect of MIF production by AML cells on MSCs recruitment will be tested using AML
conditioned medium. AML cells engineered with MIF shRNA will be used in this context. As it
cannot rule out that MSCs may also contribute to MIF production, mRNA expression will be
tested by primary normal MSC cocultured with AML cells.
It is expected that recombinant MIF will have an impact on either proliferation, cell cycle,
differentiation potential or migration of MSCs. However, MIF produced in BM microenvironment
could also modify the biology of other populations of the stem cell niche including
osteoblasts, endothelial cells or macrophages. To investigate alternative cells on which MIF
may have an impact, it will be test its effects on these populations. MSCs will be induced to
differentiate into osteoblasts, adipocytes or chondrocytes. MIF will then be added to
investigate proliferation, cell cycle and migration of populations of interest. Effect on
osteoblasts and endothelial cells will be modelized using MG63 and Huvec cell lines,
respectively.
1.9. Study population 1.9.1. Participant recruitment 300 adult patients (>18) newly diagnosed
with AML will be included in the study. 85 patients diagnosed since novembe 2015 at
Saint-Antoine Hospital will be retrospectively included in the study. 215 patients newly
diagnosed with AML at Saint-Antoine and Tours University hospitals will be prospectively
included [>100 AML patients per year are referred to Saint-Antoine hospital (>70 patients per
year) and Tours University Hospital for AML (>30 patients per year)].
1.9.2. Participant follow-up
Patient sample collection :
Only standard care blood and bone marrow samples will be used in the study :
- Blood samples for biochemistry, blood cell counts, immunophenotyping, cytogenetics,
molecular biology analyses, and tumor bank cryopreservation
- Bone marrow samples for immunophenotyping, cytogenetics, molecular biology analyses, and
tumor bank cryopreservation
Time points:
Only standard care time point samples will be collected:
- Diagnosis time point (day 0, Dx sample)
- After the first course of chemotherapy (C1 time point)
- After the second course of chemotherapy (C2 time point)
- After the third course of chemotherapy (C3 time point)
- In case of relapse or refractoriness,
- at time of diagnosis of first relapse or refractoriness (RR1)
- at time of diagnosis of second relapse or refractoriness (RR2)
- Long term remission control samples at 1, 2, 3 years after diagnosis (LTR1, 2, 3)
