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O. di Martino et al.
ATRA/bexarotene responses in diverse acute myeloid leukemias
To determine whether retinoid combination efficacy was limited to MLL-AF9 models or might extend to other leukemias, we examined the effect of combination ATRA/bexarotene on the proliferative capacity of two additional primary murine AML. The first was derived in the Timothy Ley lab using a Dnmt3a R878H knock-in allele and BM cells transduced with MSCV-FLT3-ITD virus containing GFP (Dnmt3a R878H/FLT3-IT (T Ley, unpublished observations, 2019). The second was derived in the Ross Levine lab using germline Tet2 deficiency crossed with a germline FLT3-ITD allele (Tet2-KO x FLT3-ITD).47 Both primary mouse leukemias could be grown transient- ly in methylcellulose, and we observed anti-leukemic activity of combination retinoids, with limited effects by single agents (Figure 6A and B). We examined cell growth of several human AML cell lines and primary human AML following ATRA/bexarotene treatment, noting low nM IC50 combination ATRA/bexarotene in THP1, Monomac6, OCI-AML-3, and MOLM13 (1.3±1.2, 19.2±1.7, 5.0±1.1, and 15.8±1.2, respectively), with evidence of synergy in THP1 and MOLM13, both of which contain MLL translo- cations (Figure 6C). Slightly higher IC50 values were observed for CW103-4 (8±1.1, 43±1.2, 20±1.1, and 24±1.4, respectively). We also assessed retinoid responses in a series of primary AML samples collected at diagnosis or relapse and noted evidence of response and modest synergy in samples with MLL translocations (KI_1, 7346, and 5184) (Figure 6D and E).
ATRA/bexarotene activity in vivo
To determine whether retinoid combinations could be
transitioned into in vivo activity, we first assessed the potential tolerability of the combination on normal hematopoiesis. Two cohorts of 5 wild-type mice were treated with ATRA/bexarotene by oral gavage or vehicle control. No differences were noted in the peripheral blood counts after 3 weeks of treatment (Online Supplementary Figure S7A-E), suggesting tolerance of the combination by normal hematopoiesis.
Subsequently, we tested the effect of combination retinoids on MLL-AF9 leukemia in vivo. In our first studies, we engrafted MLL-AF9 leukemia cells using sublethal irra- diation and we treated mice with slow-release ATRA/bexarotene pellets implanted subcutaneously. However, this lead to wound dehiscence and loss of the pellets (data not shown). Second, four cohorts of mice were treated with 21 days ATRA release pellets (placebo, 5 mg, 10 mg, and 25 mg, which provide an average of 0.23 mg, 0.5 mg, and 1.2 mg/day, respectively). The day after pellet implantation, all ATRA cohorts received bexarotene by oral gavage (in α-tocopherol, 50 mg/kg, which is reported to be better tolerated as a vehicle than corn oil during seri- al administration)48 (Figure 7A). However, tolerance of the combination by the mice was quite poor, as they exhibit- ed reduced activity the day after gavage, ruffled fur, weight loss, and poor wound healing, and bexarotene could be administered only every other day (qod) (Online Supplementary Figure S7F). After 21 days from pellet implantation, the mice were sacrificed and the spleen weight was analyzed. This end-point was designed to iso- late leukemic growth from confounding factors of toxicity. We observed a significant dose-dependent effect of com- bination retinoid treatment in reducing the tumor burden
(spleen weight) of treated mice compared to the control cohort (Figure 7B). Of note, the in vivo steady-state serum concentration of ATRA resulting from 10 mg 21-day release pellets is reported as 100-220 nM,49 and the 5 mg pellets did not provide a sufficient dose to inhibit leukemic growth.
Third, we suspected that some of the toxicity might be due to retinoid-induced radiation recall, a known compli- cation of retinoids,50,51 and tolerability might be improved using a micronized formula of bexarotene found in the clinical Targretin formulation.52 We found that MLL-AF9 leukemia cells engrafted consistently following IP injec- tion without irradiation conditioning, with improved fur ruffling, improved wound healing, and no weight loss (Online Supplementary Figure S7G). However, the mice again exhibited reduced activity the day after gavage and administration was again reduced to qod due to tolerabil- ity. Despite this, the combination of ATRA/bexarotene was associated with improved survival (Figure 7D and E). This study was repeated a fourth time with similar mod- est, but significant, improvement in survival and tolerabil- ity issues that persisted (data not shown). Thus, retinoid delivery to mice in the context of leukemic models remains a challenge, but is still associated with a statistical and reproducible survival advantage, although modest.
Discussion
Retinoid therapy has transformed the treatment of APL.16,17 Retinoids have been explored in different clinical trials in non-APL, and their action has repeatedly been shown, although never to the extent seen in APL.18
We previously found that natural RXRA ligands are present in mouse hematopoietic cells in vivo, that they are biased toward myeloid cells and dynamically regulated under myeloid stress (e.g., GCSF treatment).14 Consistent with those findings, in this study we found also that a malignant myeloid stressor (MLL-AF9 transformation) was associated with natural in vivo RXRA ligands. In vivo natural RXRA ligands resulted in incomplete reporter acti- vation, and pharmacologic doses of ligands could activate the reporter further under both basal conditions14 and leukemic stress (Figure 1). In contrast to these RXRA results, we have been unable to detect natural RARA lig- ands (Figure 1G).14,22
We found that MLL-AF9 cells that carried deleted Rxra and Rxrb alleles had a competitive advantage during MLL- AF9 leukemogenesis (Figure 2C and F). Whereas Notch- derived T-cell leukemias and TLS-ERG-derived ery- throleukemia were not associated with in vivo natural RXRA ligands, and also were not associated with a com- petitive advantage among cells with Rxra or Rxrb deletion (Figures 1 and 2). Thus, the presence versus absence of active natural ligands may determine whether RXR exert tumor suppressor activity.
RXR form heterodimers with a wide range of orphan nuclear receptors. Other groups have reported co-opera- tivity of RXR ligands with either RARA ligands37 or LXR ligands.53 MLL-AF9 leukemia cells appeared most sensitive to the combination of RARA and RXR ligands (Figure 3). Culture conditions influenced retinoid responses, and we observed that combinations of retinoids induced either maturation or apoptosis depending on whether the leukemia cells were grown in methylcellulose or liquid
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haematologica | 2021; 106(4)