Myelofibrosis at diagnosis is associated with the failure of treatment-free remission in CML patients

Abstract

The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence.

Keywords: CML; LSC; MSc; TFR; fibrosis; leukemic stem cells.

FIGURE 1

Clinical parameters in CML patients at initial diagnosis with different stages of BM fibrosis. (A) Proportion of CML patients with myelofibrosis (MF) in our cohort. (B) Peripheral blood (pB) blast cell count, (C) bone marrow (BM) blast cell count, (D) spleen length, (E) BM megakaryocytes (MKs), (F) pB thrombocytes and (G) pB leukocytes were assessed by the time of initial diagnoses and are represented, depending on the stage of MF. FoV: field of view.

FIGURE 2

CML transgenic mice show increased megakaryocytes upon TKI treatment. (A) Schematic representation of the mouse model and experimental set up. (B) Spleen weight at endpoint analysis. (C) BCR::ABL1 mRNA expression. Gapdh was used as housekeeping gene. (D) α-SMA staining of the sternum showing a representative example of each group. (E) Quantification of α-SMA staining. (F) FACS-immunophenotype of CD41⁺ megakaryocytes in the bone marrow. (G) FACS-immunophenotype of CD41⁺ megakaryocytes in the spleen. V: Vehicle; NIL: nilotinib; RIU: relative intensity unit. *p < 0.05, ****p < 0.0001.

FIGURE 3

Characterization of CML MSCs shows increased differentiation, but unaltered clonogenic potential. (A) MSCs were extracted from the BM and subsequently isolated via plastic adherence. (B) MSC FACS profile of MSCs in p2. (C) Healthy donor- (HD) CML-derived MSCs were analyzed for their potential to differentiate towards the adipogenic, osteogenic, or chondrogenic lineage (n = 5/5). Fold change (FC) over control of the absorbance is reported in the box plot at the bottom of panel (C). Mean FC is indicated in the graph. (D) CFU-F of HD-vs CML-derived MSC. *p < 0.05.

FIGURE 4

Expression profile of the mesenchymal CML fibrotic niche. HD-MSCs or CML-MSCs were expanded until passage 2 and RNA expression analysis was performed using a microarray. GSEA of custom gene sets for fibrosis (A) and hematopoiesis support (B) in CML vs. HD are shown. In (C), hallmark gene sets with adjusted p-value < 0.05 are shown. Specifically, Ca shows Interferon gamma response, Cb E2F targets, Cc G2M checkpoint, Cd Interferon alpha response, Ce epithelial-to-mesenchymal transition, Cf myogenesis, Cg mitotic spindle. (D) Selection of significant (adjusted p-value < 0.05) Molecular Signatures Database (MsigDB) gene sets for over-representation analysis of downregulated genes in CML vs. HD. *p < 0.05. (E) Pathway responsive genes (PROGENy) of multiple comparisons of HD-MSCs or CML-MSCs. Upregulated pathways are shown in red and downregulated ones in blue.

FIGURE 5

Presence of myelofibrosis at initial diagnosis is associated with TFR failure. The percentage of patients with or without MF that successfully reached TFR. N = 12.