Chronic myelogenous leukemia (CML) is initiated by the BCR-ABL translocation, which leads to myeloid cell expansion while allowing differentiation8–11
. Secondary translocations such as NUP98-HOXA9 or AML1-EVI1, or mutations in p53 or INK4A/ARF trigger progression through an accelerated phase to a blast crisis phase, with progressive loss of the capacity to differentiate1
. While blast crisis CML is in part more aggressive because of arrested differentiation, the pathways that underlie this arrest remain poorly understood. To determine whether CML progression may be driven by reversal of signals that regulate differentiation during normal development, we focused on Numb4
, a molecule that can be inherited differentially during asymmetric division and specify a committed fate12–16
To determine whether Numb regulates leukemia progression we utilized mouse models representing chronic phase and myeloid blast crisis CML. Chronic disease was generated by infecting hematopoietic stem cell enriched populations (c-Kit+
or KLS) with BCR-ABL and transplanting them into irradiated recipients2,3
. Myeloid blast crisis was modeled by transplanting KLS cells transduced with BCR-ABL and NUP98-HOXA96,7,17
. Using these we found that Numb was expressed at significantly lower levels in blast crisis compared to chronic phase (). The decreased expression of Numb in blast crisis suggested that keeping Numb at low levels may be essential for maintaining an immature state and that increasing its levels could trigger differentiation and inhibit disease progression. To test this possibility, hematopoietic cells were infected with BCR-ABL and NUP98-HOXA9 together with either control vector or Numb, transplanted and leukemia progression monitored. 83% of control mice developed leukemia compared to 63% of those transplanted with Numb expressing cells (). Importantly, leukemias that developed in the presence of Numb were more differentiated (), and unable to propagate disease efficiently (93% versus 20%, ) or infiltrate secondary organs (, Supplementary Fig. 1
); no signs of leukemia were detected in mice that survived (, Supplementary Figure 1
). Numb also impaired propagation of fully established leukemias and dramatically reduced the frequency of cancer stem cells (Supplementary Fig. 2
). These data show that continual repression of Numb is essential for maintenance of blast crisis CML, and that increasing the levels of Numb can inhibit disease.
Expression of Numb impairs blast crisis CML development
Since Numb can antagonize Notch signaling in several systems13,18,19
, we tested whether Numb and Notch had a reciprocal relationship in CML. Notch signaling was elevated in blast crisis CML (Supplementary Fig. 3
), and its inhibition via dnXSu(H) delivery or through conditional deletion of Rbpj paralleled the effects of Numb and led to reduced incidence and propagation of blast crisis CML (Supplementary Fig. 4
). In addition, levels of p53, another Numb target20
, were higher in Numb-expressing blast crisis CML (Supplementary Fig. 5a
). In the absence of p53, Numb was unable to impact leukemic cell growth in vivo
or in vitro
(Supplementary Fig. 5b–f
), indicating that Numb's effects are in part dependent on p53.
The observation that Numb repression was critical for maintenance of blast crisis CML led us to seek the mechanism by which Numb may be downregulated in this context. We focused on the RNA binding protein Musashi (Msi), which has been shown in the nervous system to repress Numb by binding the 3'UTR of the transcript21
. Msi was originally identified in Drosophila as a regulator of asymmetric division5,22
and its expression has been associated with stem and progenitor cells in several tissues23,24
. In the hematopoietic system we found that Msi2 was expressed at much higher levels than Msi1 (), and was particularly elevated in stem cells (). Paralleling this, Msi2 expression was 10-fold higher in the more immature blast crisis CML (); this pattern held true even in matched lineage negative fractions () suggesting that Msi2 upregulation in advanced phase is not simply a consequence of altered cellular composition. Finally, expression of Msi2 was most enriched in the lineage negative fraction of blast crisis CML (). These data indicate that Msi2 expression associates predominantly with normal hematopoietic stem cells and the most immature fraction of leukemic cells.
The RNA binding protein Musashi is highly expressed in immature normal and leukemic cells and is regulated by HoxA9
Because Msi2 and Numb were expressed in a reciprocal pattern, we tested whether Msi2 could repress Numb during leukemogenesis. Expression of Msi2 in chronic phase CML cells led to a downregulation of Numb (). Further, NUP98-HOXA9 could also activate this cascade by increasing expression of Msi2 (). Since NUP98-HOXA9 initiates transformation through HoxA9 mediated DNA binding and transcription, we tested whether HoxA9 could bind the Msi2 promoter and activate its expression directly. Chromatin immunoprecipitation revealed that HoxA9 was in fact associated with the putative HoxA9 binding element we identified at −5.7kb (). NUP98-HOXA9 expression was also able to induce Msi2 reporter activity in KLS cells (, Supplementary Fig. 6a
). These data show that Msi2 can be upregulated by NUP98-HOXA9 and subsequently contribute to blast crisis CML by repressing Numb.
To test if Msi2 is required for the development of blast crisis CML, we utilized a mouse in which the Msi2 gene was disrupted by a genetrap (Gt) vector25
(Supplementary Fig. 6a, b
). Msi2 mutant mice were viable, albeit smaller and less frequent than predicted (+/+:+/Gt:Gt/Gt=38:66:19, p=0.038), and showed a two-three fold reduction in the frequency () and absolute numbers (data not shown) of KLS cells. Additionally, the loss of Msi2 led to significantly impaired leukemia growth in vivo
(, 93% for control versus 57% for Gt/Gt).
Loss of Musashi impairs the development and propagation of blast crisis CML
To determine if inhibiting Msi2 could impact the growth of established CML, and to rule out the possibility that the reduced incidence of leukemia in Gt mutants was due to developmental defects, Msi expression was targeted using an alternate shRNA approach (Supplementary Fig. 7a
). Delivery of Msi2 shRNAs (shMsi) into established blast crisis CML cells reduced colony growth in vitro
(, Supplementary Fig. 7b–e
) and disease incidence in vivo
(). The majority of leukemias that occurred in the presence of shMsi were more differentiated (), and impaired in their ability to propagate disease (, 87% control versus 25% shMsi). These data show that Msi2 is important for establishment and continued propagation of blast crisis CML.
Finally, we examined if MSI2 was aberrantly upregulated during human leukemia progression. MSI2 was tracked in 30 patient samples from banks in Korea and the United Kingdom, and found to be expressed at significantly higher levels in blast crisis CML (). To determine if this reflected a general pattern in human CML progression, we examined expression of MSI2 and associated genes in 90 patient samples from banks in the United States26
. Microarray analysis revealed a dramatic upregulation of MSI2 in every patient during CML progression (). In addition, NUMB was downregulated in a majority of blast crisis patients (). Notably, our mouse model was driven by NUP98-HOXA9 as a second hit, whereas human blast crisis CMLs harbor a variety of secondary mutations. Since Msi2 could be regulated by HoxA9 expression in the mouse model of CML, we examined if HOXA9 was upregulated in blast crisis CML samples. The observation that a majority of patient samples had elevated levels of HOXA9 () may explain how MSI2 becomes upregulated in advanced stage disease regardless of the nature of the second hit. Notch signaling targets HES1 and TRIB2 were also elevated in a number of blast crisis patient samples (, Supplementary Fig. 8
), consistent with other independent reports27
Musashi expression is upregulated during human CML progression
Because the highest MSI2 expression was observed in blast crisis patients, where treatment outcomes are extremely poor, and because a range of expression was observed in both chronic and accelerated phase CML, we tested whether MSI2 expression correlated with outcomes after allogeneic transplantation. Patients were divided into two groups based on median expression of MSI2. Among 37 chronic phase patients with available outcomes (9 relapses) increased MSI2 expression was associated with a higher risk of relapse (hazard ratio = 4.35; 95% CI, 0.90 – 21.06, p=0.07). Additionally, among 13 accelerated phase patients with available outcomes (6 deaths and 3 relapses), increased MSI2 expression was not only associated with higher risk of relapse (all relapses occurred in the increased MSI2 group, p=0.06), but also with higher risk of death (hazard ratio = 6.76; 95% CI, 0.78 – 58.57, p=0.08). The association of MSI2 with poorer outcomes suggests that Msi2 may be an early marker of advanced CML disease.
Our work identifies the Musashi-Numb axis as an important regulator of myeloid leukemia and indicates that maintenance of the immature state is dependent on reversal of classic differentiation cues. Specifically, we find that Msi2 is upregulated and Numb downregulated as chronic phase CML progresses to blast crisis, and that modulation of this pathway can inhibit disease (). Although previous work has implicated Musashi and Numb in normal development13,14,23,24
, to our knowledge this is the first demonstration that this pathway is required for hematologic malignancy.
Our previous work showing that while BCR-ABL cannot affect the choice between asymmetric and symmetric division, NUP98-HOXA9 can trigger a bias toward symmetric renewal15
had led us to propose that regulators of asymmetric division might regulate leukemic differentiation, and could thus be targets for therapy in advanced myeloid leukemia. Our current work supports this and shows that Numb, which drives commitment and differentiation, can impair blast crisis CML establishment and propagation. It should be noted that just as Numb's influence may be mediated through p53 and/or Notch signaling12,13,20
, Musashi may act through Numb as well as other targets such as p21WAF121,28
Because blast crisis CML is uniformly resistant to current treatments, it is critical to identify new pathways that drive this aggressive disease. In that light, our work is important because it shows that specific differentiation cues associated with the Musashi-Numb cascade can unlock the differentiation potential of blast crisis CML and impair its growth. These data, together with the fact that Musashi appears to be an early marker of advanced CML, suggest that its expression could serve as a prognostic tool, and that targeting it might represent a new approach to therapy. Finally, reports of increased expression of Musashi in glioblastoma29
and decreased expression of Numb in high grade breast cancer30
raise the possibility that this pathway may also be relevant in solid cancers.