Myeloid Leukemia Stem Cells Depend on Phospholipase C Gamma 1 (Plcg1) Signaling

Several genes and signaling pathways control the fine balance between self-renewal and differentiation in hematopoietic stem cells and potentially also in leukemic stem cells (LSC). Phospholipase C family members are key mediators of calcium signaling which play an important role in differentiation and proliferation of immune cells but also contribute to malignant transformation and tumorigenesis. Plcg1 is highly expressed in hematopoietic stem- and progenitor cells and also in myeloid leukemia. Plcg1 gets activated by cell extrinsic receptor stimulation and integrates signals from the cell surface. Its influence on proliferation and differentiation of hematopoietic cells may be largely independent of other bone fide mediators of self-renewal and stem cell viability such as STAT-, MEK-ERK or AKT-signaling. To which extent Plcg1-dependent signal integration is required for function and maintenance of leukemic stem cells remained so far elusive.

Genetic inactivation of Plcg1 by RNAi in human AML cell lines led to decreased proliferative capacity. Likewise, knockdown of Plcg1 in AML1-ETO (AML1ETO9a) transformed murine LSK-cells resulted in reduced colony formation and decreased re-plating capacity.

In order to validate these findings and to investigate the impact of Plcg1 on myeloid leukemia stem cell function, we generated a conditional knockout mouse model for Plcg1 with Exons 3-5 being flanked with loxP sites. Excision of the respective sequence by activation of a Cre-recombinase resulted in complete loss of a functional protein and transcript.

LSK-cells from Plcg1^f/f and Plcg1^+/+ littermate controls were retrovirally infected with two different oncogenes: either MLL-AF9 (MA9-GFP) or AML1-ETO9a in combination with KRAS (AE9a/KRAS-GFP). Primary recipient mice were injected with GFP⁺ LSK-cells and monitored for disease development. GFP⁺ Kit⁺ cells were isolated from leukemic mice and transduced with a Cre-recombinase, followed by plating in methylcellulose.

Inactivation of Plcg1 in AE9a/KRAS transformed cells significantly reduced the number of colonies and decreased re-plating capacity to three rounds. Loss of Pclg1 in MA9 transformed LSC resulted in decreased colony numbers and colony size, however, re-plating capacity was not affected to a major extent. To assess for the requirement of Plcg1 in maintenance of fully developed leukemia, we injected equal numbers of GFP⁺ Kit⁺ cells (Plcg1^-/- or Plcg1^+/+) into sublethally irradiated secondary recipients. Inactivation of Plcg1 was highly deleterious for AE9a/KRAS induced AML-LSC and reduced disease penetrance by more than 85%. Depletion of Plcg1 in MA9 transformed cells delayed AML development and significantly prolonged survival of recipient mice. Leukemias that developed from Plcg1^-/- donors showed complete excision of Plcg1, indicating, that Plcg1 deficient leukemia can develop in an MLL-AF9 driven background. However, when transplanting MA9 transformed Plcg1^-/- or Plcg1^+/+ bone marrow cells into tertiary recipient mice, loss of Plcg1 significantly delayed disease progression and reduced disease penetrance. To quantify this loss of leukemic stem cells, we performed limiting dilution analysis using purified LSCs from diseased Plcg1^-/- or Plcg1^+/+ MA9 secondary recipient mice. LSC frequency was markedly reduced in tertiary recipients of Plcg1-depleted LSCs (1 in 78,000 Plcg1^-/- vs. 1 in 3,000 Plcg1^+/+).

Genetic inactivation of Plcg1 in LSCs derived from primary recipient mice (either MA9 or AE9a/KRAS driven AML) led to induction of differentiation as assessed by cell morphology and immunophenotyping, and this effect was more pronounced in AE9a/KRAS transformed cells.

To investigate whether transcriptional effectors of Plcg1 signaling affect the fine balance between self-renewal in MA9- and AE9a/KRAS-driven leukemia, we performed whole transcriptome analysis (RNAseq) on sorted LSCs. Ongoing analyses address the functional difference between AML-ETO and MLL-AF9 driven disease and elucidate on distinct patterns of activated gene sets depending on the oncogenic background.

Taken together, Plcg1 is required for maintenance of myeloid leukemia stem cells. Understanding of its relevance in LSC biology and function may offer the opportunity to develop this relevant signaling node as a target structure in AML.