Page 102 - Haematologica Vol. 109 - July 2024

ARTICLE - CD47 inhibition is augmented with TLR3 agonism H.E. Ramsey et al.
comparing them to naïve NSGS mice. In the BM, the more tumor permissive M2 phenotype was dominant in PDX and undifferentiated M0 phenotypic macrophages were more common in native NSGS mice (Online Supplementary Figure S3A). These findings imply that the presence of AML blasts within the BM affects the relative number of macrophages, and polarizes those resident macrophages toward a tu- mor-permissive phenotype.
TL3 agonism leads to the expansion of F4/80 macrophages in the bone marrow, spleen and blood of treated mice
Given to the historical application of IFN-γ in myeloid malignancies, and the role IFN-γ plays in macrophage dif- ferentiation, IFN-γ was proposed as potential therapeutic to artificially increase macrophage populations within the BM.26-28 However, IFN-γ has only been shown to increase or activate macrophages with the addition of LPS.29-31 Poly(I:C) is a TLR3 agonist which mimics viral double-stranded RNA in a sterile immune response known to stimulate macro- phage differentiation and polarization to M1 macrophages.18 Systemic administration of poly(I:C) in a TLR3-deficient mouse resulted in significant reductions in both macro- phage activation and cytokine production compared to TLR3 wild-type.32 Additionally, poly(I:C) has synergized previously with CD47 blockade in colorectal cancer mod- els.33 As variations of poly(I:C) are in use in over 13 clinical trials currently as an immunoadjuvant, we adapted use of
this TLR3 agonist in our murine models.34-37 Administering poly(I:C) over a 2-week period revealed significant shifts in macrophage phenotype, most notably the additional presence or increase in M1 macrophage populations (Fig- ure 5A). Furthermore, the ratio of M1/M2 macrophages changed significantly between the treatment time points in these naïve NSGS mice, with dramatic shifts being noted after 2 weeks of poly(I:C) (Figure 5B). Additionally, levels of macrophages in of poly(I:C)-treated mice increased while receiving treatment (Figure 5C).
TLR3 agonism potentiates CD47i by increasing active macrophages in a patient-derived xenograft model
Given the response of naïve NSGS mice to TLR3 agonism via poly(I:C) treatment, we attempted to increase BM mac- rophage populations to augment the effect of ALX90 in PDX models. In a AML PDX (18-12-001) mice were treated with ALX90 and or poly(I:C) to illustrate effects of TLR3 stimulation on CD47 inhibition. We found that poly(I:C) augmented the use of ALX90, but had negligible effects on the BM chimerism when used as a single agent (Online Supplementary Figure S3B). Neither PDX patient samples (18-12-001, 18-10-009, an additional sample 21-12-019) nor cell lines (MV-4-11, MOLM-13) were affected by poly(I:C) alone, suggesting that poly(I:C) is not inherently toxic to blast cells (Online Supplementary Figure S4A). In order to illustrate if addition of TLR3 agonism could improve upon triplet therapy (Figure 2), we transplanted PDX 18-10-009
AB
C
Figure 4. Analysis of acute myeloid leukemia patient-derived xenograft resident murine macrophage populations. (A-C) Mu- rine macrophage (F4/80) levels were measured by flow cytom- etry in both patient-derived xenograft (PDX) models under ALX treatment, and within a naïve untreated NSGS mouse in periph- eral blood, splenic (SPL) and bone marrow (BM) compartments. *P<0.05, **P<0.01 and ***P<0.001.
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