Fig 2: HSF1 is dispensable for stressed hematopoiesis.a Kaplan–Meier survival curves of Hsf1fl/fl (Control, Con) or Hsf1fl/flVav-Cre+ (Hsf1KO) mice treated with weekly doses of 5-FU (150 mg/kg, IP, Con n = 7 mice; Hsf1KO, n = 6 mice). b HSC (defined as Lin-cKit+Sca1+CD150+ CD48-) numbers of Con or Hsf1KO mice at different time points after sublethal (6.5 Gy) radiation (Con, n = 3 mice at each time points, Hsf1KO, n = 3 mice at Day 0 and 10, n = 5 mice at Day 30 and n = 4 at Day 45). c Percentages of HSCs in Con or Hsf1KO mice (3 mice/group) treated with 35 µg of LPS twice (48 h apart) and analyzed 24 h after the second injection. d Donor chimerisms of mice serially transplanted with control (Con) or Hsf1KO HSCs (primary, 500 cells/mouse, secondary and tertiary BMT, 2 × 106 BM cells from primary or secondary recipients. e Frequencies of CD3+ T cells, B220+ B cells, Mac1+Gr1- monocytes and Mac1+Gr1+ granulocytes in primary, secondary, and tertiary recipient mice transplanted with Con or Hsf1KO KLS cells. d, e n = 4/group for primary and secondary BMT, and n = 5/group for tertiary BMT. In (b–e), data are presented as mean values ± SEM. Source data are provided as a Source Data file.

Fig 3: HSF1 is required for the initiation and maintenance of MLL-AF9–induced AML.a Survival curve of mice receiving MLL-AF9 transduced LSK (Lin–cKit+Sca1+) cells from Hsf1fl/fl (Con) or Hsf1fl/flVav-Cre + (Hsf1KO) mice (n = 5 mice/group). Log-rank test, *p = 0.0021. b Scheme of serial plating for establishing immortalized leukemia cells. c MLL-AF9 Hsf1fl/flcreER leukemia cell numbers in the presence or absence (vehicle, Con) of 4-hydroxytamoxifen (the active metabolite of tamoxifen, 4-OHT, 200 nM). 1 × 103 leukemia cells were plated and counted 3–4 days later (n = 6 independent replicates). *p = 2.5 × 10-5. d Colony numbers formed in the presence or absence of 4-OHT (n = 8 independent replicates). *p = 7.3 × 10-6. e 500 cells from vehicle (Con) or 4-OHT treated colony assay (d) were replated without further 4-OHT treatment (n = 6 independent replicates). *p = 4.24 × 10-7. f Scheme of establishment of MLL-AF9–induced AML and delivery of tamoxifen to induce Hsf1 deletion. g Survival curve of mice receiving MLL-AF9–transduced LSK cells from Hsf1+/+creER or Hsf1fl/flcreER mice with or without Tam treatment (n = 8 mice/group). Log-rank test, *p = 0.0002. h Frequencies of LSCs in MLL-AF9 Hsf1fl/flcreER leukemic mice with or without Tam treatment (n = 4 independent replicates). *p = 2.2 × 10-5, **p = 0.017. i Frequencies of CD11b+ leukemia cells in MLL-AF9 Hsf1fl/flcreER leukemic mice with or without Tam treatment (n = 3 independent replicates). *p = 2.79 × 10-5. In (c–e, h, i), two-tailed t test was used, data are presented as mean values ± SEM. j Wright-Giemsa staining of LSC-derived colony forming cells with or without 4-OHT treatment (n = 3 independent replicates). Note that Tam treatment induced LSC differentiation with reduced nuclear to cytoplasmic ratio. Scale bar, 5 µM. k Survival curve of mice receiving LSCs from full-blown MLL-AF9 Hsf1fl/flcreER leukemic mice treated with or without Tam (n = 5 mice/group). Log-rank test, *p = 0.0019. l Survival curve of mice receiving LSCs from full-blown MLL-AF9 Hsf1fl/flcreER leukemic mice treated with two cycles of Tam (n = 5 mice/group). Log-rank test, *p = 0.0031. Source data are provided as a Source Data file.

Fig 4: HSF1 is critical for maintenance of LSC self-renewal.a Engraftment of three different primary AML LSCs in the presence or absence of CRISPR-mediated HSF1KD (started 4 mice/AML). *p = 0.0404, **p = 0.0003, ***p = 0.0006. b ATP production in control or HSF1 knockdown AML cells (post-transplantation from a) measured by Seahorse assay (n = 3 independent replicates). AML-a: *p = 0.0046. **p = 0.0349; AML-b: *p = 0.0201. **p = 0.0124; AML-c: *p = 0.0325. **p = 0.0033; c The expression of HSP90, SDHC, and HSF1 proteins in SISU-102 treated mouse MLL-AF9 cells and the human AML cell lines MV4–11 and NOLM-1 (n = 3 independent replicates). ACTIN is used as a load control. d Cell growth of murine MLL-AF9 cells (*p = 1.6 × 10-5, **p = 3.1 × 10-5, ***p = 6.1 × 10-5) and human AML cell line MV4–11 (*p = 4.8 × 10-3, **p = 2.3 × 10-4, ***p = 1.6 × 10-4) and NOLM-1 (*p = 6.3 × 10-4, **p = 3.01 × 10-5, ***p = 6.38 × 10-5) in the presence of absence of SISU-102 (n = 3 independent replicates). e OCR in Hsf1fl/flcreER LSCs (*p = 8.87 × 10-31, **p = 1.09 × 10-7) or human AML cell line MV4–11 (*p = 4.89 × 10-13, **p = 4.97 × 10-12) and NOLM-1 (*p = 1.19 × 10-18, **p = 0.00028) treated with or without SISU-102 (n = 8 independent replicates). f Engraftment of human AML cell line MV4–11 (n = 5 mice/group, *p = 9.8 × 10-5) or three different primary AML cells in the presence or absence of SISU-102. AML-d: *p = 1.8 × 10-5, n = 4 mice/group, and AML-e: *p = 8.98 × 10-6, Con n = 4 mice, SISU-102, n = 5 mice; AML-f: *p = 0.0011, Con n = 5 mice, SISU-102, n = 4 mice. g Engraftment of human BM CD34+ HSPCs treated with (n = 4 mice) or without (n = 3 mice) SISU-102 (5 mg/kg, IP, daily). In (a, b, d–f), two-tailed t test was used, data are presented as mean values ± SEM. Source data are provided as a Source Data file.

Fig 5: Expression of HSF1 might serve as a marker to monitor AML status.a HSF1 H-score index in AML samples at diagnosis, remission, or relapse (n = 39 primary AML samples). Box plots show median and first/third quartiles. ANOVA with LSD post-hoc analysis was used, *p = 0.0001. b–f Representative pictures showing 2 different HSF1 staining patterns in AML patients. b–c Pattern 1, HSF1 staining at remission (b) and follow up (c). d–f Pattern 2, HSF1 staining at remission (d), follow up (e) and relapse (f). Note that (b) and (d) are 2 weeks post chemotherapy, BM shows 5–10% of cellularity. The insets in (b) and (d), are HSF1 staining at AML diagnosis. Scale bar, 20 µM. Source data are provided as a Source Data file.