High Levels of the Adhesion Molecule CD44 on Leukemic Cells Generate Acute Myeloid Leukemia Relapse After Withdrawal of the Initial Transforming Event

Abstract

3154 Multiple genetic hits are detected in patients with acute myeloid leukemia (AML). To investigate this further, we developed a tetracycline inducible mouse model of AML, where the initial transforming event, overexpression of HOXA10, can be eliminated. Continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients showed that in established leukemias, ∼80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous since ∼20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10 induced overexpression (HOXA10OFF). Since the withdrawal of the initial transforming event can be made upon demand, we have been able to ask what co-operating events are essential to maintain growth of leukemic cells as overexpression of HOXA10 is removed. Intrinsic genetic activation of several proto-oncogenes was observed in leukemic cells resistant to inactivation of the initial transformation event. We have identified a frequent increase in the activation of the proto-oncogenes JUN, FOS and EGR1 in relapsed leukemia where overexpression of HOXA10 has been withdrawn (HOXA10OFF versus HOXA10ON conditions). In order to further investigate if another possible mechanism is involved in leukemia, upon withdrawal of the primary oncogenic event, we performed proteomic analysis using mass spectrometry. Interestingly, we observed that upon removal of the primary event, leukemia that continued to grow produced high levels of several proteins involved in cell-cell and cell-matrix interactions. Among these proteins, CD44 is expressed on the cell surface and participates in cell transmigration and is an important target, since this surface glycoprotein mediates cell adhesion, migration and homing of hematopoietic cancer cells. To determine whether an increase in CD44 is a key mechanism by which LICs are resistant, we performed a functional test by FACS sorting leukemic cells generated in primary donors and transplanted 20,000 cells expressing different levels of the CD44 surface marker (CD44low, CD44medium and CD44high) in the tail vein of lethally irradiated secondary recipient mice fed doxycycline or ciproxine. When we monitored mice for occurrence of leukemia, outgrowth of leukemic cells was not dependant on the CD44 protein level on the HOXA10ON (doxycycline) condition. Consistent with this, onset of leukemia was not delayed for mice transplanted with CD44low leukemic cells. When mice were fed with ciproxine to turn off HOXA10 overexpression, all mice injected with CD44high leukemic cells developed leukemia, whereas all mice injected with CD44low leukemic cells remained healthy. In conclusion, we confirmed that withdrawal of the initial HOXA10 oncogene promotes the outgrowth of LICs expressing high levels of CD44. This study suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia. Here we demonstrate the highly aggressive nature of LICs expressing high levels of CD44 and conversely, show the impaired outgrowth of LICs expressing low levels of this surface marker. In conclusion, our murine model of inducible HOXA10 expression recapitulates many of the features of human AML and is helpful to analyse the “oncogene addiction” and unravel the basic mechanisms involved in initiation and maintenance of leukemia, and to study whether adhesion molecules expressed on the surface of leukemic cells are important factors for leukemic relapse in the microenvironmental niches of the bone marrow. Our findings support the notion that cell intrinsic genetic events are not the only factors causing leukemic relapse, but suggest that host-dependant extrinsic factors in the bone marrow niche may also play a fundamental role in the mechanism mediating leukemic relapse. Disclosures: No relevant conflicts of interest to declare.

Transglutaminase2 Expression in Acute Myeloid Leukemia: Association with Adhesion Molecule Expression and Leukemic Blast Motility

“…In support of the concept that TG2 promotes the expression of integrins and adhesion markers, thereby increasing leukemic-stromal interaction is the observation that both the white blood cell count and the absolute peripheral blood blast count were inversely correlated with TG2 levels. This TG2 induced stabilization of cell-cell and cell-niche interaction could also be responsible for the reported relationship between CD44 expression and the relapse of AML [25]. Thus there is a positively reinforced pathway in many cases of AML studied here, with respect to extracellular matrix association.…”

Transglutaminase 2 expression in acute myeloid leukemia: Association with adhesion molecule expression and leukemic blast motility

“…In support of the concept that TG2 promotes the expression of integrins and adhesion markers, thereby increasing leukemic—stromal interaction is the observation that both the white blood cell count and the absolute peripheral blood blast count were inversely correlated with TG2 levels. This TG2 induced stabilization of cell–cell and cell–niche interaction could also be responsible for the reported relationship between CD44 expression and the relapse of AML . Thus there is a positively reinforced pathway in many cases of AML studied here, with respect to extracellular matrix association.…”

Follistatin is a novel therapeutic target and biomarker in FLT 3/ ITD acute myeloid leukemia

“…Proteomics analyses in both FST344‐ and FST317 ‐overexpressing ML‐2 cells showed consistent increases in protein expression of Cathepsin G (CTSG), TATA‐box binding protein‐associated factor 15 (TAF15), CD44, RNA binding motif protein 39 (RBM39), hematological and neurological expressed 1 (HN1), and Poly(C)‐binding protein 2 (PCBP2) (; ) of which TAF15 (Ballarino et al , ), CD44 (Quere et al , ), HN1 (Zhang et al , ), and PCBP2 (Han et al , ) have been associated with cancer initiation and progression. On the other hand, proteins associated with nonsense‐mediated decay of mRNA (NMD) were significantly decreased in FST344 ‐overexpressing ML‐2 cells ().…”