Leukemia Stem Cell Marker CD123 (IL-3R alpha) Predicts Minimal Residual Disease and Relapse, Providing a Valid Target For SL-101 In Acute Myeloid Leukemia With FLT3-ITD Mutations

Leukemia stem cells (LSC) play critical role in initiation and progression of acute myeloid leukemia (AML). CD123 (IL-3R alpha) is one of the established LSC markers, and the frequency of CD123⁺ LSCs at diagnosis is associated with response to treatment (Vergez F et al, Haematologica 2011). However, it remains unclear whether CD123⁺ LSCs contribute to minimal residual disease (MRD) and disease recurrence.

In this study, we first analyzed expression of CD123 on CD34⁺ blast cells in a cohort of 95 newly diagnosed AML patients (pts) treated with induction regimens between 02/2011 and 10/2012. In these pts, standard MRD assessment was routinely performed at the time of complete remission (CR) as described (Ravandi et al, Leukemia 2012). CD123 was highly expressed in diagnostic BM samples (67%, range 26.8%-91.7%). Higher expression of CD123⁺ cells on blasts and within CD34⁺ stem/progenitor cells correlated with poor prognosis cytogenetics (p=0.016 and 0.034) and unmutated NPM1 (p<0.001 and p=0.003, respectively). Altogether, 62.1% (59/95) pts achieved negative MRD, which in a multivariate analysis strongly correlated with longer time to relapse (HR: 0.71; 95%CI: 0.58-0.87; p<0.001). A higher proportion of CD123⁺CD34⁺ cells in remission marrows (ratio of %CD34⁺CD123⁺/%CD34⁺ greater than 0.83) was associated with higher risk to remain MRD-positive in both univariate (OR, 0.11-0.96, p=0.043) and multivariate Logistic Regression models (p=0.027). Although higher frequencies of CD34⁺CD123⁺ blasts significantly correlated with shorter time to relapse in the univariate Cox proportional hazard model (p=0.037), only white blood count, FLT3-ITD mutation and time to achieve MRD remained significant in a multivariate analysis.

We next analyzed expression of CD123 in enriched CD34⁺CD38^-CD99⁺ LSC fraction (Jan et al, Sci Transl Med. 2012) in 10 newly diagnosed AML BM samples. As controls, we used 3 normal bone marrow (NBM) samples. Cell surface characteristics and signaling pathways activation were defined by the time-of-flight mass cytometry (CyTOF) (Bendall et al, Science. 2011) using 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). The frequency of CD123⁺ cells within CD34⁺CD38^-CD99⁺ LSCs was significantly higher in AML samples at diagnosis (median 78.1%, range 38.4%-96.3%) compared to NBM (median 25.1%, range 22.8%-39.6%; p<0.001). FLT3-mutated AML expressed higher levels of CD123 within LSCs (median 90.9%, range 72.9%-96.3%) compared to FLT3-wt (median 69.5%, range 38.4%-73.5%; p=0.013). Multiple intracellular signaling pathways were highly activated in CD123⁺ LSCs in AML with FLT3-ITD compared to NBM stem cells, including p-4EBP1, p-NF¦ÊB, p-AKT, p-mTOR and p-ERK (Figure 1). This data indicate that targeting CD123-expressing LSC may have anti-leukemia effects in AML, especially those with FLT3 mutations.

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To this end, we tested anti-leukemia activity of SL-101, a novel targeted therapy directed to CD123, in AML cell lines. SL-101 is an anti-CD123 scFv antibody conjugate. CD123 is highly expressed in AML cell lines (n=12) except for U937 and HL-60 cells (median 75.2%, range 0.9%-99.8%). SL101-induced killing strongly correlated with CD123 expression (r=-0.8220, p=0.001), suggesting that SL101 induces target-specific cell killing at low nanomolar range (median IC₅₀ 10.9 nM, range 0.1 nM-249.9 nM). We next analyzed the combined effects of SL101 and FLT3 inhibitor sorafenib in three FLT3-ITD cell lines. Strikingly, exogenous IL-3 significantly protected FLT3 mutant cells from sorafenib-induced cell killing in MV4;11 (p<0.01) and MOLM13 cells (p<0.001) but not in MOLM14 cells. In turn, combination with SL-101 revoked IL-3 mediated cytoprotection in MV4;11 (p<0.001) and MOLM13 cells (p<0.01).

In conclusion, expression of CD123 on CD34⁺ blast cells is associated with persistent MRD in newly diagnosed AML. Multiple signaling pathways are activated in these CD123⁺ LSCs, supporting the feasibility of targeting CD123 to eliminate AML LSCs. SL-101 induces apoptotic cell death in CD123⁺ AML cell lines at low nanomolar concentrations and abrogates IL-3 mediated cytoprotection in FLT3-mutant cells. Ongoing studies are focusing on the anti-leukemia efficacy of SL-101 in primitive LSCs in primary AML samples, especially those with FLT3-ITD.