University of Groningen
Modeling of MLL-AF9-rearranged pediatric leukemia Carretta, Marco
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Carretta, M. (2018). Modeling of MLL-AF9-rearranged pediatric leukemia: Identification of mechanisms and potential targets. University of Groningen.
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CHAPTER
5
The cell of maintenance in MLL-rearranged B-ALL is multipotent
and retains myeloid potential while MLL-rearranged AML is
maintained by myeloid-restricted LSCs
M. Carretta1#, A. Brouwers-Vos1, E. de Bont2, E.Vellenga1, and J.J. Schuringa1*
Affiliations:
1Department of Experimental Hematology, Cancer Research
Center Groningen (CRCG), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2Division of Pediatric Oncology/Hematology,
Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
(Manuscript in preparation)
Abstract
Although at low frequency, switches between lymphoid and myeloid lineages can occur during treatment or during relapse of pediatric leukemia patients bearing MLL-translocations. The mechanisms behind these phenomena have remained elusive, but published studies suggest that both intrinsic and environmental cues may inspire changes in cell fate decisions of leukemic stem cell clones. Here, by making use of a lentiviral model in which we introduce MLL-AF9 in human CB-derived CD34+ stem/progenitor cells as well as primary MLL-rearranged pediatric patient samples we aimed to model lineage switching in vitro and in vivo in xenograft mice. Our data show that upon environmental changes it is possible to convert CD19+ B-ALL cells to
a complete myeloid phenotype, while CD33+ AML cells bearing the AF9 or MLL-AF4 translocation are not able to switch to a lymphoid phenotype. Clonality studies and RNA-sequencing performed before and after the switch confirmed the same clonal origin and a complete switch from a lymphoid to myeloid gene signature. Our observations support findings obtained in MLL-rearranged pediatric leukemia patients and point to the notion that B-ALL is maintained by multipotent leukemic stem cells (LSCs) that retain myeloid potential, while AML appears to be maintained by more myeloid-restricted LSCs. Understanding the plasticity of LSCs is of crucial importance to determine the pathogenesis and disease progression of MLL-rearranged leukemia and for the design of new therapeutic strategies addressing these rare cases.
Introduction
Chromosomal translocations involving the mixed lineage leukemia (MLL) gene define a unique group of leukemias, generally associated with poor prognosis 1,2. These
translocations are found in 10% of adult cases and over 70% of pediatric patients, where they can be associated with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or biphenotypic acute leukemia (BAL) 3. Over 50 different translocation fusion partners have been described in literature, but only a limited subset accounts for most cases 4. Among the most frequent MLL rearrangements in
pediatric patients, the MLL-AF9 fusion is found in 18% of the total ALL cases and in 33% of the total AML cases, while MLL-AF4 is found in 34% of ALL cases and only in rare cases of AML and BAL 3,4.
Rare cases of conversion of the lymphoid and myeloid leukemic cell lineage during the course of the disease or during relapse have been reported in literature. According to published data, lineage switching is almost exclusively observed in pediatric patients rather than in adults, and it occurs most frequently from the lymphoid to the myeloid lineage 5-9. Several hypotheses have been proposed to explain the molecular basis of the lineage switch, but the actual mechanisms remain to be discovered. Possible explanations are the emergence of new therapy-related clones or the emergence of therapy-insensitive clones of a different lineage already present at low frequencies at the first diagnosis. Another explanation might be the reprogramming of a multipotent leukemic stem cell that in response to intrinsic or environmental cues can change the lineage of its progeny 10. Although these complete lineage switches occur infrequently, it is relevant to evaluate whether the leukemic blasts originate from the same leukemic clone or whether new clones emerge as a consequence of the treatment.
Since our current knowledge on lineage switches is largely based upon clinical studies, in the present work we aimed to model these switches in an in vitro and in vivo setting to gain more insights into the underlying mechanisms. Previously, we established in vitro models that faithfully recapitulate the AML and B-ALL phenotypes observed in patients by lentiviral transduction of the MLL-AF9 fusion into CD34+ cells from fetal cord blood (CB) and adult bone marrow (BM) cells 11. Here, by using this MLL-AF9 CB model and primary MLL-AF4 patient cells we show that it is possible to convert CD19+ B-ALL cells to a complete myeloid phenotype upon changing the environmental cues. Clonality studies and RNA-sequencing performed before and after the switch confirmed the same clonal origin and a complete switch from a lymphoid to myeloid gene signature. CD33+ AML cells bearing MLL-AF9 or MLL-AF4 translocation instead were incapable to switch to a lymphoid phenotype upon change of environmental cues. Our data support the existence of a multipotent cell of maintenance in B-ALL that retains myeloid potential, while AML appears to be maintained by more myeloid-restricted leukemic stem cells.
Material and Methods Primary cell isolations
Neonatal CB samples were obtained after informed consent from healthy full-term pregnancies from the obstetrics departments of the University Medical Centre in Groningen (UMCG) and Martini Hospital Groningen. BM mononuclear cells from untreated patients were used after informed consent and protocol approval by the Medical Ethical Committee of the UMCG, in accordance with the Declaration of Helsinki. After ficoll separation of mononuclear cells, CD34+ cells were enriched using a magnetically activated cell-sorting CD34 progenitor kit or automatically by using auto Macs (Miltenyi Biotech) as described previously 12 and cryopreserved until further use.
Lentiviral transductions
CB CD34+ cells were pre-stimulated and transduced as described previously 12,13.
One round of transduction was performed and cells were harvested at day 2 after transduction. For MLL-AF9 transformation of CB CD34+ cells UMG LV6 MLL-AF9 lentiviral vectors were used 14.
Cell Culture
CB transduced MLL-AF9 cells in the liquid and MS5 myeloid co-culture experiments were grown in Gartner’s medium consisting of αMEM (Fisher Scientific Europe, Emergo, The Netherlands) supplemented with 12.5% heat-inactivated fetal calf serum (Lonza, Leusden, The Netherlands), 12.5% heat-inactivated horse serum (Invitrogen, Breda, The Netherlands), 1% penicillin and streptomycin, 2mM glutamine (all from PAA Laboratories), 57.2 µM β-mercaptoethanol (Merck Sharp & Dohme BV,
Haarlem, The Netherlands) and 1 mM hydrocortisone (Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands). CB MLL-AF9 myeloid restricted cultures were supplemented with 20 ng/ml IL-3, SCF and FLT3-L (R&D Systems). Lymphoid permissive co-cultures contained the same components as the myeloid cultures with the exception of hydrocortisone and horse serum but with the presence of 50 µg/ml ascorbic acid (Sigma). In lymphoid CB MLL-AF9 co-cultures IL-3 was replaced with 10 ng/ml IL-7 (R&D Systems). AML cells in myeloid restricted cultures were supplemented with 20 ng/ml IL-3, SCF, FLT3-L, granulocyte colony-stimulating factor (Rhone-Poulenc Rorer, Amstelveen, The Netherlands), and thrombopoietin (Kirin, Tokyo, Japan) as previously described13. All the cultures were kept at 37°C and 5% CO2.
FACS analysis
Cells were incubated with primary antibodies at 4˚C for 30 min. For blocking non-specific binding to Fc receptors, cells were blocked with mouse and human anti-Fc antibodies for 5 min at 4˚C prior to the staining with primary antibodies. All FACS analyses were performed on MACSQuant Analyzer 10 (Miltenyi Biotech) or LSR II Flow Cytometer (BD) and data was analysed using Flow Jo (Tree Star, Inc.). Cells were sorted on a MoFlo (Beckman Coulter). The following antibodies (all from Biolegend) were used: CD45 (HI30), CD19 (HIB19), CD15 (W6D3), CD11b (ICRF44), CD20 (2H7), CD14 (HCD14), CD33 (WM53).
RNA sequencing
Total RNA was isolated using the RNeasy micro kit from Qiagen (Venlo, The Netherlands) according to the manufacturer’s recommendations. An initial quality
check and RNA quantification of the samples was performed by capillary electrophoresis using the LabChip GX (Perkin Elmer). With 50ng mRNA sequence libraries were generated using the Lexogen Quantseq 3’ prep kit (Lexogen GmbH) according to the manufacturer’s recommendations. The obtained cDNA fragment libraries were sequenced on an Illumina NextSeq500 using default parameters (single read). All the bioinformatics were performed on the Strand Avadis NGS (v3.0) software (Strand Life Sciences Pvt.Ltd). Raw sequence quality was checked for GC content, base quality and composition using FASTQC and StrandNGS. Quality trimmed reads were aligned to build a Human HG19 (UCSC) transcriptome. Ensembl genes and transcripts (2014.01.02) were used as gene annotation database. Quantified reads were normalization using the DESeq package. Reads with failed vendor QC, reads with average quality less than 24, reads with mapping quality below 50 and reads with length less than 20 were all filtered out.
MLL-AF4 PCR
Total RNA was isolated using the RNeasy micro kit from QIAGEN (Venlo, the Netherlands) according to the manufacturer’s recommendations. RNA (250 ng) was reverse transcribed with MMLV reverse transcriptase (Biorad). cDNA was realtime amplified in iQ SYBR Green supermix (Biorad) with the MYIQ thermocycler (Biorad). Single and Nested PCR were performed according to the UMCG guidelines for diagnosis of MLL-AF4 translocations (protocol available on request).
Ligation-mediated-PCR (LM-PCR)
Results and discussion
Lymphoid transformed CB MLL-AF9 cells can convert to the myeloid lineage Previously, we have described the development of an in vitro MLL-AF9 model where CB transduced cells are grown on a stromal feeder layer of mouse BM origin (MS5) or human BM origin (MSCs) 11,17. In myeloid ‘restricted’ conditions, the cells are
grown in the presence of hydrocortisone, horse serum and cytokines such as IL3, while the lymphoid ‘permissive’ condition is without hydrocortisone, horse serum, and supplied with IL7. Depending on the culture conditions, CD34+ transduced cells of
neonatal origin can readily immortalize along the myeloid and lymphoid lineage 11,17. First, we evaluated whether lymphoid transformed CB MLLAF9 cells could be converted to the myeloid lineage by changing the culture conditions. At start, these lymphoid transformed MLL-AF9 cells expressed CD19, but were negative for CD33, CD14, CD11b or CD15 (Fig. 1A). When these cells were grown under lymphoid-permissive conditions for 1-3 weeks, no change in the FACS phenotype was observed (Fig. 1B). In striking contrast, when these cells were grown under myeloid-restricted culture conditions a gradual loss of CD19 expression and a gain of CD33 expression was observed (Fig. 1B). Already after one week, over 90% of the cells converted to the myeloid lineage and after three weeks 100% of the cells were CD33+ with the co-expression of myeloid markers such as CD14 and CD11b. (Fig. 1B). The cumulative expansion under myeloid conditions was even higher than under lymphoid conditions (Fig. 1C).
Next, we performed clonality studies to understand whether the myeloid clones arose from the initial CD19+ lymphoid clone, or whether a new clone preferentially grew out
under the myeloid-restricted culture conditions. Ligation-mediated PCR (LM-PCR) showed that the initial cultures were dominated by one clone (Fig. 1D). After 3 weeks
of myeloid culture conditions a complete switch from lymphoid to myeloid phenotype was seen, and again the same clone dominated the culture as shown by LM-PCRs (Fig. 1D). Together, these data indicate that the LSCs that maintain the B-ALL culture retain multi-lineage potential and are capable of generating myeloid progeny depending on extrinsic cues.
Figure 1. Lymphoid-transformed CB MLL-AF9 cells can convert to the myeloid lineage. A) CB CD34+ cells were transduced with MLL-AF9 and were transformed
along the B-lymphoid lineage. B) B-ALL CB MLL-AF9 cells were cultured under lymphoid or myeloid conditions for a period of 3 weeks. FACS plots for CD33, CD19, CD11b, CD14 and CD15 of the suspensions cells are shown. C) Total cumulative expansion of CB MLL-AF9 cells under myeloid or lymphoid culture conditions is shown. D) LM-PCRs were performed to study clonality of MLL-AF9 B-ALL cultures at the start, after three weeks grown under lymphoid conditions, or after three weeks grown under myeloid conditions. The internal control band is indicate by an arrow, the red asterisk represent a viral integration site which belongs to the dominant clone.
Myeloid-transformed CB MLL-AF9 cells and primary AML samples cannot convert to the lymphoid lineage
In the clinic, lineage conversion in MLL-related leukemia is found to be almost exclusively associated with conversion from the lymphoid to myeloid lineage 5,8. Yet,
we wished to evaluate whether we could reprogram myeloid-transformed CB MLL-AF9 cells to the lymphoid lineage in our in vitro model systems. We transduced CB CD34+ cells with MLL-AF9, cultured them for a period of 2 weeks under myeloid conditions, after which we switched the culture conditions from myeloid to lymphoid. These cells were still able to convert to a lymphoid phenotype (data not shown), indicating that after the initial 2-week culture period the multipotent stem cells were still present. Next, CB MLL-AF9 cells were first kept under myeloid culture conditions for 12 weeks. These cells were completely CD33+ and also co-expressed CD14 and CD11b (Fig. 2A). Remarkably, these cells were no longer able to give raise to lymphoid progeny when grown under lymphoid-permissive culture conditions (Fig. 2A), opposite to what was observed for the lymphoid to myeloid conversion (Fig. 1). No differences were observed in growth kinetics when week-12 myeloid transformed MLL-AF9 cells were consecutively grown for a period of 4 weeks under myeloid or lymphoid growth conditions (Fig. 2B).
We next moved to an in vivo setting, where we xenotransplanted cells in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. These mice are known to be rather lymphoid-biased, and lymphoid B-ALL is easily generated upon engraftment of MLL-AF9-transduced CB CD34+ cells 11,16,17. We engrafted in vitro immortalized myeloid MLL-AF9 CB CD34+ cells in NSG mice, but no lymphoid leukemia could be generated (data not shown and 17), indicating that also under these lymphoid-permissive in vivo
In adult MLL-rearranged leukemia, patient’s lineage switches are hardly ever observed. In fact, we previously showed that, in contrast to fetal CD34+ cells isolated from CB, the immortalization of CD34+ transduced BM cells isolated from adults donors is skewed towards the myeloid lineage, in line with what is observed in patients. This clearly indicates that the differences in ontogeny of the cell of origin from which the leukemia arises has an impact on the lineage of the progeny 11. Although the exact underlying molecular mechanisms are still under investigation, it is quite plausible that difference in the epigenetic landscape of the cell of origin account for the observed differences in lineage output. Here we show that myeloid transformed BM MLL-AF9 cells can strongly proliferate under myeloid conditions, but are indeed incapable of converting to the lymphoid lineage (Fig. 2C). The same was observed when MLL-AF9 AML cells isolated from an adult patient were used, since again we were not able to convert them to the lymphoid lineage (Fig. 2D), although their proliferation did not seem to be affected (data not shown). These data clearly confirm the hypothesis that MLL-rearranged AML is maintained by more committed LSCs or leukemic-GMPs that appear to have only retained myeloid potential and have lost lymphoid potential.
Figure 2. Myeloid-transformed CB MLL-AF9 cells and primary AML samples cannot convert to the lymphoid lineage. A-B) CB CD34+ cells were transduced with MLL-AF9 and transformed along the myeloid lineage for a period of 12 weeks (top panels). After that, cells were cultured under lymphoid conditions for a consecutive period of 4 weeks (lower panels). FACS plots for CD33, CD19, CD11b, CD14 and CD15 of the suspensions cells are shown in A and total cumulative expansion in B. C) Adult BM CD34+ cells were transduced with MLL-AF9 and transformed along the myeloid lineage for a period of 5 weeks (top panels). After that, cells were cultured under lymphoid conditions for a consecutive period of 4 weeks (lower panels). FACS plots for CD33, CD19, CD11b, CD14 and CD15 of the suspensions cells are shown. D) MLL-AF9 cells isolated from the BM of a primary adult AML patient were cultured in lymphoid conditions for a period of 4 weeks. FACS plots for CD33, CD19, CD11b, CD14 and CD15 of the suspensions cells are shown.
Primary neonatal B-ALL cells can convert to the myeloid lineage
Lastly, we wished to extend our findings also to primary pediatric MLL-rearranged leukemias. For these experiments, we used cells derived from patients (n=2): sample #1 displaying a CD19+ lymphoid phenotype with some CD14+ cells (Fig. 3A, top
panels) and sample #2 presenting with a biphenotypic acute leukemia with the presence of individual CD19+ and CD33+ clones (Fig. 3C, top panels).
Upon 3 weeks of culture under myeloid conditions, all CD19+ cells from patient #1 were able to switch to a myeloid phenotype with co-expression of markers such as CD33, CD14, CD11b and CD15 (Fig. 3A). Under lymphoid-permissive conditions where both myeloid and lymphoid cells can proliferate, we noted that a small population of CD19+ cells was retained but also a large population of myeloid CD33+ cells appeared (Fig. 3A). In fact, when we compared proliferation rates of cells grown under myeloid versus lymphoid conditions, cells grown under lymphoid conditions displayed the slowest growth rate (Fig. 3B). This might be related to the fact that primary lymphoid patient cells are more difficult to expand under in vitro conditions, and explains the preferential outgrowth of myeloid cells even under the lymphoid-permissive growth conditions. Nevertheless, it is clear that predominantly CD19+ lymphoid cells from this MLL-AF4 pediatric B-ALL patient were able to give rise to myeloid CD33+, CD11b+ and CD14+ cells.
Next, since pediatric patient #2 presented two distinct CD33 and CD19 cells population at diagnosis, we wished to determine whether in particular the CD19+ cells would be able to undergo a lineage shift as we observed earlier in our CB models. Therefore, CD33+ and CD19+ cells were sorted and both were grown under myeloid or lymphoid conditions. As expected, CD33 cells could be expanded under both myeloid-restricted and lymphoid-permissive conditions but did not convert to the
lymphoid lineage and remained CD33+ (Figure 3C). Interestingly, when CD19+ cells were grown under myeloid-restricted conditions, a strong outgrowth of CD33+ cells was seen (Fig. 3C) and these cells displayed a clear myeloid morphology on cytospins (Fig. 4A). Like was seen in B-ALL patient #1, when CD19+ cells were
grown under lymphoid-permissive conditions both lymphoid as well as myeloid cells were detected. Importantly, we performed PCRs to detect whether the MLL-AF4 translocation would still be present after a 3-week culture period under myeloid-restricted conditions, and in all cases, so also in the case where CD19+-sorted cells gave rise to exclusively CD33+ cells, the MLL-AF4 translocation was still detected
(Fig. 3D). These data again strongly suggest that also in MLL-rearranged B-ALL pediatric patients CD19+ clones are maintained by multipotent LSCs that retain myeloid potential.
Figure 3. Primary pediatric MLL-rearranged B-ALL cells can convert to the myeloid lineage. A) CD34+ cells isolated from a pediatric MLL-AF4 B-ALL patient #1
were analyzed by flow before in vitro culturing (top panels) or after in vitro culturing under myeloid-restricted or lymphoid-permissive conditions for a consecutive period of 3 weeks (lower panels). FACS plots for CD33, CD19, CD11b, CD14 and CD15 of the suspensions cells at week 3 are shown. B) Cumulative expansion under myeloid-restricted or lymphoid-permissive conditions. C) CD34+ cells isolated from a pediatric MLL-AF4 B-ALL patient #2 were analyzed by flow before in vitro culturing (top panels). CD19+ and CD33+ cells were sorted as indicated and were cultured under
myeloid-restricted or lymphoid-permissive conditions for a consecutive period of 3 weeks, and also bulk cells without prior sorting were cultured under these conditions as indicated (lower panels). FACS plots for CD33, CD19, CD11b, CD14 and CD15 of the suspensions cells at week 3 are shown. D) Bulk cells, CD19+-sorted cells or
CD33+-sorted cells were grown under myeloid-restricted conditions and analyzed for MLL-AF4 by PCR.
We wished to further substantiate these findings and study the identity of lymphoid to myeloid converted clones by performing genome-wide transcriptome studies by RNAseq. As shown in Figure 3C, CD33+ and CD19+ cells of patient #2 were sorted and these populations were grown under either myeloid-restricted or lymphoid-permissive conditions for three weeks. FACS phenotypes and cytospins confirmed the myeloid phenotype of the expanded populations (Fig.4A). RNA was isolated from the bulk blast population at the start (RNAseq #1), from the CD19+ population directly
after sort (RNAseq #2), from the CD19+-sorted population grown for 3 weeks under myeloid-restricted conditions (RNAseq #3) and from the CD33+-sorted population grown for three week under lymphoid-permissive conditions (RNAseq #4) (Fig. 4A). Unsupervised cluster analysis revealed that the bulk population and the CD19+ sorted population clustered together, while the CD19-sorted population that was converted to a CD33+ myeloid clone mostly resembled the in vitro expanded CD33-sorted cells (Fig. 4B). These data clearly indicate that the myeloid-converted cells have adopted a complete myeloid transcriptional program, which was further underscored by GSEA analyses that revealed that the CD19 to CD33 converted cells lost B-cell signatures (Fig. 4C) and had gained myeloid signatures (Fig.4D). Individual expression profiles of B-cell specific and myeloid specific genes are shown in Fig. 4E. In contrast, target genes specific for MLL-rearranged leukemias were rather comparable in all 4 populations, further confirming the presence of MLL-AF4-drivene gene expression programs (Fig. 4F).
Figure 4 Myeloid-converted B-ALL cells acquire a complete myeloid transcriptional program A) CD33+ and CD19+ cells of patient #2 were sorted and grown under either myeloid-restricted or lymphoid-permissive conditions. Facs phenotypes and cytospins of expanded populations after three weeks are shown. B) Unsupervised cluster analysis show the bulk population and the CD19+ sorted
population clustering together, while the CD19-sorted population converted to CD33+
myeloid clone resembled the in vitro expanded CD33-sorted cells. E-F) GSEA showing the CD19 to CD33 converted cells lost B-cell signatures (4E) and had
gained myeloid signatures (4F). G) Individual expression profiles of B-cell specific and Myeloid specific genes are shown H) Target genes specific for MLL-rearranged, further confirming the presence of MLL-AF4-drivene gene expression programs
In conclusion, our data show that is possible to recapitulate the lineage switch observed in MLL-rearranged leukemia (MLL-r) in our in vitro systems. Conversion of the leukemic cell lineage is almost exclusively associated with MLL-r pediatric leukemia and in the clinic most cases involve the conversion of B-ALL to AML. Accordingly, upon changes of environmental conditions in our co-culture system, we were able to convert CD19+ B-ALL to a complete myeloid phenotype in a CB transduced MLLAF9 model as well as primary MLL-AF4 pediatric patient sample. Clonality studies and RNA-sequencing performed before and after the switch confirmed the same clonal origin and a complete switch from a lymphoid to myeloid gene signature. Conversely, CD33+ AML cells driven by MLL-AF9 were able to
convert to a lymphoid phenotype only within the first two weeks of culture, while cells cultured for longer times did not generate CD19+. Conversion of CD19-CD33+ to
lymphoid lineage was not achieved also in primary MLL-AF4 pediatric cells and adult MLL-AF9 cells.
Although in the clinic specific MLL fusion genes are predominately associated with both myeloid and lymphoid leukemia and thus suggesting an instructive role of the MLL fusion partner gene, it became progressively clearer that LSC retain plasticity and the lineage fate is also influenced by microenvironmental cues. The susceptibility of MLL-r cells to extrinsic cues has been previously shown in various in vivo models
16,18,19. For example, conversion from the lymphoid to myelomonocytic leukemia was
observed upon injection of human CB cells retrovirally transduced with MLL-AF9 and transformed along the lymphoid lineage in NOD/SCID (NS) mice transgenic for the
observed in normal NS mice. Furthermore, CD19-CD33+ cells did not generate CD19+ cells in cell culture but only in some cases produced both CD33+ AML and CD19+ ALL in mice 19.
These findings highlight the importance of the microenvironmental signals in fate decision of MLL-r leukemia, but also point to the established notion that these leukemias can originate from different cells. Previous studies in mice have shown that both hematopoietic stem cells (HSCs) and lineage-restricted progenitors such as Granulocyte Macrophage Progenitor (GMP) can become LSC when targeted by MLL fusion gene. Moreover, fusion proteins have also been shown to confer the ability to re-install a self-renewal program 20,21,22.
We therefore propose a model where if the initial oncogenic event occurs at the level of HSCs or multipotent progenitors (CD34+), the immortalization by MLL-AF9 can lead to ALL, AML or BAL. The LSC, which maintain these leukemias, is multipotent and can be influenced by microenvironmental cues. Experimental and clinical evidence supports the notion that MLL-rearranged ALL is maintained by a more immature LSC, which can always be converted to AML upon changes of microenvironmental signals (Figure 5, scenario 1). AML driven by MLL-AF9 instead can also be maintained by a multipotent LSC, but as the disease progresses in time the AML appears to be maintained by more committed LSCs, which lose their ability to convert to the lymphoid lineage (Figure 5, scenario II). An alternative scenario is that the initial oncogenic event occurs at the level of more committed hematopoietic cells such as GMPs. In this case, the LSC is natively lineage-restricted and the immortalization by MLL-AF9 can lead only to AML, excluding the possibility of conversion to the lymphoid lineage (Figure 5, scenario III). Interestingly, MLL-AF9-positive AML patients can be divided into EVI1high and EVI1low subgroups whereby
the EVI1high group is associated with an inferior outcome 23. Since EVI1 expression is high in HSCs and much lower in GMPs it is plausible that differences in the cell of origin as highlighted in Figure 5 might account for these differences in the observed clinical features.
Lineage switching offer an interesting example of the lineage heterogeneity observed in acute leukemias and as this phenomenon correlates with particularly bad prognosis further studies are needed to improve therapeutic options. Progress in the available sequencing technologies, in vitro and in vivo models and knowledge of leukemia pathogenesis will help to understand the exact mechanisms driving the lineage switching during treatment or relapse.
Figure 5 Proposed model of lineage fate determination in MLL-AF9 leukemia The initial oncogenic event can occur at the level of the HSCs, and the immortalization by MLL-AF9 can lead to ALL, AML or a mixture of the two (BAL). The LSC which maintain these leukemias is multipotent and can be influenced by microenvironmental cues. AML driven by MLL-AF9 can be maintained by a multipotent LSC, but as the disease progress in time they appeared to be maintained by a more committed LSC which loses its ability to convert to the lymphoid lineage. The initial oncogenic event can occur also at the level of more committed hematopoietic cells such as GMPs. In this case, the LSC is lineage-restricted and the immortalization by MLL-AF9 can lead only to AML, excluding the possibility of conversion to the lymphoid lineage.
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