Modulation of Leukemic Stem Cell Self-Renewal and Cell Fate Decisions by Inhibition of Hedgehog Signalling in Human Acute Lymphoblastic Leukemia (ALL).

Abstract 2578

The Hedgehog (Hh) pathway plays a functional role in embryonic development and promotes tumorigenesis in a diversity of cancers. Constitutive activation of Smo, an essential component of the Hh pathway, augments stem cell number and accelerates disease in BCR-ABL positive CML, whereas loss of Smo depletes CML stem cells by inhibition of self-renewal. Phase I clinical trials using Hh inhibitors have started in BCR-ABL pos ALL and CML. The role of Hh signalling on stem cell behaviour in BCR-ABL neg ALL has not yet been examined. The phenotype of leukemic stem cells (LSCs) and the target cells for transformation in ALL are controversial, but only a small subpopulation of cells seem to act as LSCs. These cells may be the most relevant targets for treatment regimens for compounds that interfere with self-renewal programs and that provide promising therapeutic options for improving treatment of adult ALL.

Aims of the study are characterization of different genetically and phenotipically defined ALLs, using our twelve patient derived long term cultures (LTCs), according to their biologic behaviour including leukemia initiating capacity, assessment of the impact of Hh inhibition on proliferation, apoptosis and clonogenic capacity and LSC function and dissection of the role of different components of the Hh signalling pathway on cell fate decisions by means of single cell video microscopy. These studies are anticipated to yield information on the therapeutic potential of modulation of Hh signalling in both BCR-ABL pos and neg ALL and the potential value of combination therapy.

As models of BCR-ABL pos and neg leukemias we used serum-, cytokine- and stroma-free long term cultures of primary ALL blasts. Clonogenic growth of ALL cells was assessed in semi solid methylcellulose based media. Cell subpopulations were isolated on the basis of CD20, CD34 and CD38 expression via FACS based sorting (BD FACS Aria). Cell proliferation was measured using XTT assays and Annexin V and 7 AAD FACS staining were used to quantitate apoptosis. Quantitative RT PCR of Hh signalling pathway components using predeveloped Taqman assays (Applied). Single cell video tracking to determine cell fate decisions was performed as previously described (Rieger et all, Science 2009), adapted to facilitate the analysis of ALL LTCs. Two novel Smo inhibitors being currently in clinical testing, LDE225 and BMS833923 were kindly provided by Novartis and Bristol Myers Squibb.

Results: The expression pattern of surface markers varied profoundly between the different LTCs studied. In preliminary experiments designed to identify functionally distinct subpopulations of long term cultured ALL blasts, cells were isolated to greater than 90% purity based on CD20, CD34 and CD38 expression. With the exception of CD34 positive cells, the surface marker distribution rapidly reverted to an identical pattern as determined prior to culture in three cell lines studied. In two ALL LTCs, CD34 expression was associated with slower proliferation. All three cell lines displayed clonogenic capacity ranging from 0,25% to 8% and are able to engraft in NSG mice.

Analysis of Hh Signalling in ALL LTCs by RT PCR demonstrated expression of Shh, Ptch, Smo, and the transcription factors Gli 1 + 3, indicating active Hh signalling in ALL. Interestingly the transcription factor Gli 2 was not expressed, the functional relevance of which remains as yet unclear. The Hh inhibitors LDE225 and BMS833923 (0,01μM to 5μM) showed no dose dependent effect on inhibition of proliferation or induction of apoptosis in ALL LTCs.

In conclusion we found evidence of Hh activation in both BCR-ABL pos and neg LTC ALL cells. No impact of Smo inhibition on proliferation and apoptosis was observed in response to the Smo inhibitors LDE225 and BMS833923, consistent with the hypothesis that Hh signalling in these cells may affect primarily self-renewal mechanisms. Single cell imaging of ALL LTCs has been successfully established for up to nine days of culture and will be applied to the testing of Hh modulation on cell fate decisions.