Fig 2: AURKA PROTAC cocktail induces AML regression in a xenograft mouse model and primary blasts. A) Nude mice bearing KG1A xenografts were intraperitoneally injected with dAurA383, dAurA450, and cocktail (30 µmol kg−1 day−1). The tumor volume from 1 to 14 days is plotted versus time. B) Left panel, tumors resected from the mice in each group are shown. Right panel: statistical analysis of the tumor weight. C) The surface CD34 and CD11b of dissected tumors were analyzed by FACS assays. D) The indicated proteins of dissected tumors were detected by Western blot. E) The body weight of the mice were measured and plotted against time. F) Plasma ALT, AST, and BUN of the mice were measured. The dotted lines display the normal reference values of BALB/c nude mice in Charles River Laboratories. G) AURKA expression was detected by Western blot in AML primary blasts treated with AURKA PROTACs (1 × 10−6 m) for 6 h. H) Cell apoptosis was analyzed in AML primary blasts treated with AURKA PROTACs (1 × 10−6 m) for 48 h. Statistics, significance: one‐way ANOVA with Bonferroni correction (A,B,C,F); ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: The combination of gDE7 and 1MT improves mice survival depending on their administration regimen. (A–D) The data represent the average of two independent experiments (n=5, total n=10). (A, B) Survival curves. Data were analyzed by Kaplan-Meyer test. (A) Two mice from gDE7 group and from gDE7 + DL-1MT survived until day 60. (B) Two, one, and four mice from gDE7, gDE7 + D-1MT, and gDE7 + DL-1MT groups survived until day 60, respectively. (C, D) Tumor-free mouse curves. Data were analyzed by ANOVA test* p <0.05. (E) Gating strategy for immune cell analyses in the tumor microenvironment, evaluated at day 21 after the tumor engraftment. Doublets were initially excluded by FSC-H versus FSC-W parameters, followed by SSC-H versus SSC-W parameters. Cells were gated by the expression of CD45+ and successively analyzed for: CD8+, followed by CD8+ IFN-g+; CD4+, followed by CD25+ and FoxP3+ CD25+ (T regulatory cells); CD11chigh MCH-IIhigh (dendritic cells); and Gr1high CD11b+. (F) Frequency of CD45+ cells. (G) Frequency of Gr1high CD11b+ cells. (H) Frequency of CD11chigh MCH-IIhigh cells. (I) Frequency of CD8+ T cells. (J) Frequency of E7-specific CD8+ IFN-g+/CD8+ cells. (#) p<0.05 represent the statistical significance of stimulated (red dots) versus non-stimulated (white dots) cells inside each experimental group. (K) Frequency of CD4+ T cells. (L) Frequency of FoxP3+ CD25+ CD4+ cells. Data representative of two independently performed experiments (n=6). Statistical significance: (*) p<0.05, (**) p<0.01 by ANOVA. (#) p<0.05 (###) p<0.001 represents the ANOVA statistical significance of stimulated (red dots) versus non-stimulated (white dots) cells inside each experimental group.

Fig 4: TLR4 on hematopoietic cells is critical for oxaliplatin-induced mechanical hyperalgesiaa) Exogenous administration of LPS through gastric gavage reversed the effect of gut microbiota eradication. Mice were fed with antibiotic water (abx) or regular water (H2O), followed by oxaliplatin injection. LPS (3mg/Kg) or normal saline was administered on days of oxaliplatin injection and twice weekly afterwards. *p<0.05, abx,oxaliplatin; LPS gavage vs. abx, oxaliplatin; saline gavage, N=7 each group. b) Toll-like receptor 4 (TLR4) knockout mice did not develop oxaliplatin-induced mechanical hyperalgesia. TLR4 knockout (TLR4−/−) or littermate heterozygous (TLR 4+/−) mice were treated with oxaliplatin or saline and were examined at indicated time points for hindpaw mechanical withdrawal threshold (HWT) (N=6 each group). * p<0.05 WT, oxaliplatin vs. all other groups. c–d) Generation of bone marrow (BM) chimeras. c) Flow chart for BM chimeric mice generation. Recipients were irradiated 500 Rad × 2 followed by bone marrow cells injection. Donor bone marrow cells were derived from TLR4−/− or WT mice, and were injected to WT or TLR4−/− recipients, using a cross-over study design. CD 45.1 and CD45.2 congenic markers were used to distinguish between donor-derived and recipient-derived hematopoietic cells. d) Confirmation of successful generation of bone marrow chimera. Fourteen weeks after BM transplantation, DRG samples were collected and stained for CD45.1 and CD45.2 congenic markers. Contour plots were gated on CD11b+ and CD3− cells. Each panel represents 6 independent stainings. e) TLR4 on hematopoietic cells is critical for oxaliplatin-induced mechanical hyperalgesia. BM chimeric mice generated as shown in c–d) were treated with oxaliplatin (N=6 each group). In TLR4−/− to WT group, hematopoietic cells were from TLR4−/− donors. These mice were protected from oxaliplatin-induced mechanical hyperalgesia despite the presence of TLR4 on host-derived radio-resistant cells. *p<0.05 TLR4−/− to WT vs. WT to WT. In contrast, in WT to TLR4−/− group, hematopoietic cells were from WT donors, these mice developed oxaliplatin-induced mechanical hyperalgesia despite the absence of TLR4 on host-derived radio-resistant cells. ** p> 0.05 WT to TLR4−/− vs. WT to WT.

Fig 5: Kat2a loss facilitates development of Idh1R132H preleukemia.(A) Diagram of Idh1R132H (Idh1 mut) and Kat2afl/fl mouse crosses to generate Idh1 mut Kat2a HET and Idh1 mut Kat2aNULL cells used in preleukemia studies. WT, wild-type. (B) CFC assays of Idh1 mut Kat2a HET and NULL BM cells 4 weeks after polyinosinic:polycytidylic acid (pIpC) treatment. Mean ± SD, n = 3. (C) CFC assays of Idh1 mut Kat2a HET and NULL BM cells 20 weeks after pIpC treatment; mean ± SD, n = 3. (D) Quantification of GMP-like BM cells obtained from Idh1 mut CD45.2+ grafts; mean ± SD, n = 3 irradiated recipients (CD45.1). (E) Representative flow cytometry plots of BM cells in (D). Top: Idh1 mut Kat2a HET. Bottom: Idh1 mut Kat2aNULL. KLS, Lin-Kit+Sca1+. (F) Serial replating CFC assays of Idh1 mut BM grafts. Mean ± SD, n = 3 Idh1 mut Kat2a HET and NULL and n = 2 Idh1 mut leukemia. (G) Flow cytometry of colonies in (F). Left: representative plots. Right: Kit+Mac1− progenitor quantification. Mean ± SD, n = 3. All analyses two-tailed t test, *P < 0.05.