A Novel TNF-Alpha Antibody Based Therapeutic Approach to Target Leukemic Stem Cells in Bcr-Abl Disease

Chronic myeloid leukemia (CML) reflects the biology of a stem cell driven neoplasm. The cytogenetic marker t(9;22) gives rise to Bcr-Abl protein that activates various signaling pathways resulting in increased proliferation and differentiation, protection from apoptosis and altered adhesion properties of CML cells. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, more than one third of newly diagnosed CML patients develops primary or secondary resistance mostly due to Bcr-Abl mutations or intolerance to TKIs. Moreover, even in patients without mutations, mechanisms such as low persisting Bcr-Abl levels in patients on TKI and/or a lack of oncogene addiction have been described to induce CML stem cell persistence.

We have previously demonstrated Bcr-Abl independent stem cell persistence using inducible SCLtTA/Bcr-Abl mice (BLOOD 2010, 115:3185; BLOOD 2012, 119:1501), and recent data suggest an important role for TNF-alpha in MPN stem cell persistence (BLOOD 2011, 118:6392), including CML (BLOOD 2013, 112:3335). Therefore, we have now interrogated our mouse model for a novel stem cell directed therapeutic approach, using pharmacologic inhibition of TNF-alpha.

Analysis of TNF-alpha expression by qRT-PCR in Bcr-Abl infected 32Dcl3 and Ba/F3 cells revealed elevated cytokine expression upon Bcr-Abl transduction. Imatinib treatment of 32Dcl3:Bcr-Abl cells reverted elevated TNF-alpha levels suggesting that TNF-alpha upregulation is kinase mediated in this cell line. ChIP-Seq of TKI treated vs non-treated 32Dcl3:Bcr-Abl cells showed H3K9 acetylation of the TNF-alpha promoter in untreated cells, which decreased upon Bcr-Abl inhibition. Subsequently, we analyzed TNF-alpha expression in lin^-;Sca-1⁺;c-kit⁺ (LSK) cells isolated from SCLtTA/Bcr-Abl mice that had been induced to express Bcr-Abl for 3 weeks. Leukemic LSK cells showed again significantly increased TNF-alpha expression. Interestingly, TNF-alpha RNA levels were not altered in total bone marrow (BM) cells isolated from transplanted leukemic Bcr-Abl mice compared to controls, suggesting a stem cell specific mechanism regulating TNF-alpha expression in vivo. Next, we tested the therapeutic effect of TNF-alpha inhibition combined with TKI therapy. We applied the chimeric antibody infliximab that blocks soluble as well as membrane-bound TNF-alpha. We transplanted 1.5x10⁵ wildtype (wt) or Bcr-Abl BM cells expressing CD45.1 into 10 Gy irradiated CD45.2⁺ wt recipients (wt n=4; Bcr-Abl n=24) and allowed these cells to engraft and expand for 2 weeks. We then either treated Bcr-Abl transplanted mice with nilotinib alone (50mg/kg, daily) or combined the TKI therapy with infliximab (10mg/kg, weekly i.v.). As controls, we either treated Bcr-Abl transplanted mice with vehicle alone (daily) or together with IgG (10mg/kg, i.v., weekly). Wt recipients remained untreated. After 2.5 weeks of therapy, we sacrificed all mice for analyses. Spleen weight of vehicle and vehicle+IgG treated mice were increased compared to nilotinib and nilotinib+infliximab treated recipients (Table 1).

Table 1: Analyses of mice that were subjected to TNF-alpha and Bcr-Abl inhibition

	wt	Bcr-Abl
		vehicle	vehicle + IgG	nilotinib	nilotinib + infliximab
spleen weight (mg)	73 ± 5	274 ± 63	280 ± 55	125 ± 5	185 ± 31
% of lin- cells	4.8 ± 1.1	31.1 ± 11.0	25.0 ± 14.1	3.4 ± 1.0	3.1 ± 1.4
% of LSK/CD45.1+ cells	5.7 ± 0.7	41.2 ± 5.9	31.6 ± 11.7	20.3 ± 10.8	7.2 ± 3.4

FACS analyses s that lin^- cells were significantly elevated in vehicle and vehicle+IgG treated Bcr-Abl recipients compared to wt mice. Nilotinib and nilotinib+infliximab therapy decreased lin^- cells to 3.4% and 3.1% respectively. As expected, leukemic CD45.1⁺ LSK cells were significantly increased in vehicle (7-fold) and vehicle+IgG (5.5-fold) treated mice compared to CD45.1⁺ wt LSK cells. Nilotinib treatment decreased the elevated levels of CD45.1⁺ LSK cells compared to wt controls to 3.6-fold but combining TKI+infliximab decreased the leukemic stem cell population further to 1.3-fold difference compared to wt mice.

In conclusion, we show that TNF-alpha expression is elevated in Bcr-Abl positive leukemic stem cells and this cell population is significantly reduced by combining TKI therapy with an anti TNF-alpha approach. Our data reveal that this combinational therapy is a powerful tool to target the TKI resistant stem cell population in CML.