Page 121 - 2020_07-Haematologica-web

Transforming activities of the NUP98-KMT2A fusion
Supplementary Table S5) and that seen in our cohort of pri- mary-induced, non-irradiated iNUP98-KMT2A mice on doxycycline (median latency 26 vs. 80 weeks; P=0.0003, log-rank test, n=6) (Online Supplementary Figure 3A). All symptomatic mice had extensive multi-organ infiltration by leukemic blasts, which were visible on peripheral blood smears as well as in the BM and kidneys (Figure 3G). Collectively, these data show that expression of iNUP98-KMT2A leads to an MDS-like disease and in some cases to transplantable AML in mice.
iNUP98-KMT2A+ acute myeloid leukemia cells do not express the HoxA-B-C gene cluster and are resistant to compounds targeting the KMT2A-menin interaction
In contrast to other NUP98 fusions, primary patients’ NUP98-KMT2A AML cells were shown to express reduced levels of the HOXA-B-C gene cluster.19 Likewise, leukemic blasts from diseased iNUP98-KMT2A mice gen- erally expressed very low levels of HoxA5, HoxA9, HoxA10, HoxB4, HoxB6, HoxC6 and HoxC9 mRNA com- pared to normal BM cells or to leukemic blasts trans- formed by retroviral KMT2A-ENL (rKMT2A-ENL) or the rKMT2A-AF9 fusion genes (Figure 5A).24
The lack of KMT2A exon 1 encoding for the very N-ter- minus, which mediates the menin/LEDGF interaction, predicts that cells carrying the NUP98-KMT2A fusion protein would be resistant to small molecule menin inhibitors. However, if leukemic transformation by NUP98 fusions depends on KMT2A, as suggested by recent studies,14,25 NUP98-KMT2A leukemic blasts might be susceptible to inhibition by small molecules targeting critical KMT2A functional interactions. To address this question, we exposed cells from two leukemic iNUP98- KMT2A mice (“M1” & “M3”) to different doses of the small molecule menin inhibitor (MI-2-2) and to a bro- modomain inhibitor blocking BET-family proteins includ- ing BRD4 (JQ1) previously shown to efficiently block KMT2A and KMT2A-fusion controlled transcription.26 As shown in Figure 5B, MI-2-2 (3-12 μM) did not impair growth of iNUP98-KMT2A leukemic blasts but at 6 μM and 12 μM induced a G1 cycle arrest in leukemic cells expressing the rKMT2A-AF9 fusion (P<0.0001, two-way ANOVA, n=2), with a concomitant decrease in the pro- portion of cells in the S-phase (P<0.0001, two-way ANOVA, n=2) and in the G2/M-phase (6 μM: P=0.0246; 12 μM: P=0.0144, two-way ANOVA, n=2). Exposure of iNUP98-KMT2A cells to low (0.05-0.5 μM) doses of JQ1 did not induce cell cycle arrest or significant cytotoxicity as seen in rKMT2A-AF9 cells (Figure 5C), but increased the fraction of cells in the G1 phase of the cycle, suggest- ing alternative transforming mechanisms of NUP98- KMT2A compatible with a defective cell cycle checkpoint control.
Discussion
Inv(11)(p15q23) has been reported in a heterogeneous group of human hematologic malignancies including MDS, AML, peripheral T-cell lymphoma, childhood acute lymphoblastic acute leukemia, myeloma and hairy cell leukemia.20 In the majority of the myeloid cases, inv(11)(p15q23) was the sole cytogenetic abnormality, suggesting a role as a leukemogenic driver. Expression of a NUP98-KMT2A fusion gene has so far been reported in two AML patients with inv(11)(p15q23).19 Detailed molecular work-up of additional patients is needed to delineate the epidemiology of this rare entity. Notably, inv(11)(p15q23) was also found in some solid cancers but never analyzed in more detail.20 To determine the trans- forming potential of the NUP98-KMT2A fusion gene we developed inducible transgenic mice. This approach avoids some of the drawbacks of retroviral gene transfer such as cooperating integration events or transduction bias of early myeloid progenitor cells. In addition, the large size of the NUP98-KMT2A fusion ORF would clear-
Expression of iNUP98-KMT2A results in aberrant cell cycle progression and escape from senescence
In the presence of doxycycline (1 μg/mL), ex vivo prolif- eration of Lin- iNUP98-KMT2A BM cells was significantly impaired in liquid culture containing cytokines (murine stem cell factor, interleukin-6, and interleukin-3 (Online Supplementary Figure S3B). Expression of iNUP98-KMT2A was verified on day 6 of the culture (Online Supplementary Figure S3C). However, iNUP98-KMT2A expression did not significantly alter the clonogenic growth of BM cells in methylcellulose containing doxycycline (Online Supplementary Figure S3D). Cell cycle analysis of Lin- iNUP98-KMT2A BM cells challenged with doxycycline in vitro revealed an increase in the number of cells in G1 phase (P=0.033, unpaired t-test, n=3) at the expense of G0 and G2/M phases (P=0.049, unpaired t-test, n=3) (Online Supplementary Figure S3E), similar to what was observed in LSK from iNUP98-KMT2A mice on doxycycline (Figure 2C).
To further explore the impact of iNUP98-KMT2A expression on cell cycle regulation we established MEF. We first verified iNUP98-KMT2A expression in the MEF (Figure 4A). We then determined cell cycle progression of iNUP98-KMT2A MEF on doxycycline and found accumu- lation of the cells in the G1 phase (P=0.081, unpaired t- test, n=3) with a significant reduction of the percentage of cells in the G2/M phase (P=0.029, unpaired t-test, n=3) (Figure 4B). Initially, both iNUP98-KMT2A and WT MEF grew at similar rates; however, upon serial propagation of the cells we observed reduced growth of WT MEF with signs of senescence (visualized by X-gal staining for senescence-associated β-galactosidase activity) after 10- 13 passages (Figure 4C, Online Supplementary Figure S3F, G). In contrast, iNUP98-KMT2A MEF continued to grow at an increased rate (P=0.0156, Wilcoxon matched-pairs signed rank test, n=2) up to, and beyond, passage 40. To understand how iNUP98-KMT2A MEF escape senes- cence, we compared the expression of 84 genes related to cell cycle regulation and senescence using a commercial array-based reverse transcription PCR assay. Combining two independent experiments revealed no significant changes in gene expression at an early time-point (pas- sage 1) (Figure 4D) while the levels of expression of eight genes were significantly reduced in iNUP98-KMT2A MEF relative to WT MEF at later passages (passage 10-13) (Figure 4E).
Genes that were found to be dysregulated in late-pas- sage iNUP98-KMT2A MEF samples were further analyzed in iNUP98-KMT2A HSPC, which had been exposed to doxycycline in vitro for 48 h. Expression patterns observed in MEF for Sirt1, Rbl2, Twist1, Prkcd, Vim, and Tert were found to be similar in iNUP98-KMT2A HSPC, demon- strating common patterns of gene regulation in MEF and primary iNUP98-KMT2A cells (Figure 4F).
haematologica | 2020; 105(7)
1863