Journal: iScience

Article Title: Selective inhibition of ALDH1A3 impedes breast cancer growth and metastasis by blocking ALDH1A3-driven transcriptional programs

doi: 10.1016/j.isci.2026.114863

Figure Lengend Snippet: CLM296 inhibits ALDH1A3-mediated Aldefluor fluorescence and is specific for ALDH1A3 (A) Western blots showing ALDH1A3 and ALDH1A1 protein expression in MDA-MB-231, MDA-MB-468, and HCC1806 cells with respective ALDH1A3 or ALDH1A1 OE or ALDH1A3 KD clones. (B) The effect of CLM296 on ALDH1A3-mediated Aldefluor fluorescence performed on MDA-MB-231 vector control or ALDH1A3 OE cells ( n = 4), MDA-MB-468 with ALDH1A3 KD or shControl ( n = 3), and HCC1806 with ALDH1A3 KD or shControl ( n = 3) when treated with increasing concentrations of CLM296 (0.01 nM–100 μM). Significance determined by one-way ANOVA followed by multiple comparisons post-test and p value < 0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. Each point represents a separate replicate ( n = 3) and error bars represent standard deviation (SD). (C) IC 50 calculations for CLM296 against ALDH1A3 activity based on Aldefluor assay data. IC 50 was calculated using GraphPad Prism 9 using log(inhibitor) vs. response – variable slope (four parameters). Each point represents the mean of three separate n and error bars represent SD. (D) The effect of CLM296 on ALDH1A1-mediated Aldefluor fluorescence in MDA-MB-231 cells with or without ALDH1A1 OE when treated with increasing concentrations of CLM296 (1 nM–100 μM, n = 3). Significance determined by one-way ANOVA followed by multiple comparisons post-test and p value <0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. Each point represents a separate replicate ( n = 3) and error bars represent SD. NT, no treatment

Article Snippet: Anti-ALDH1A3 mouse IgG monoclonal primary antibody , Origene Technologies , Cat # CF502842 ; RRID; AB_3738370.

Techniques: Fluorescence, Western Blot, Expressing, Clone Assay, Plasmid Preparation, Control, Standard Deviation, Activity Assay

Journal: iScience

Article Title: Selective inhibition of ALDH1A3 impedes breast cancer growth and metastasis by blocking ALDH1A3-driven transcriptional programs

doi: 10.1016/j.isci.2026.114863

Figure Lengend Snippet: CLM296 specifically impedes ALDH1A3-specific gene expression in TNBC cell lines (A) Heatmap of non-hierarchical clustering of RNAseq data in MDA-MB-231 vector control and ALDH1A3 OE cells treated with or without 100 nM CLM296. (B–D) Volcano plot to identify differentially expressed genes in MDA-MB-231 cells with (B) ALDH1A3 OE untreated versus vector control untreated samples, (C) vector control CLM296-treated samples versus vector control untreated samples, and (D) ALDH1A3 OE CLM296-treated samples versus ALDH1A3 OE untreated samples. Each point represents a gene, with the x axis showing log 2 FC and the y axis representing -log 10 FDR. Non-significant genes (FDR ≥0.05) are shown in gray. The dashed line represents log 2 FC of 0.6. Key differentially expressed genes of interest are labeled. (E) RT-qPCR of ALDH1A3-inducible genes in MDA-MB-231, MDA-MB-468, and HCC1806 cells with respective ALDH1A3 OE or ALDH1A3 KD clones treated with increasing concentrations of CLM296 (0 nM, 1 nM, 10 nM, 100 nM, all n = 4). Significance determined by one-way ANOVA followed by multiple comparisons post-test and p value < 0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. Each point represents a separate n and error bars represent SD. NT, no treatment.

Article Snippet: Anti-ALDH1A3 mouse IgG monoclonal primary antibody , Origene Technologies , Cat # CF502842 ; RRID; AB_3738370.

Techniques: Gene Expression, RNA sequencing, Plasmid Preparation, Control, Labeling, Quantitative RT-PCR, Clone Assay

Journal: iScience

Article Title: Selective inhibition of ALDH1A3 impedes breast cancer growth and metastasis by blocking ALDH1A3-driven transcriptional programs

doi: 10.1016/j.isci.2026.114863

Figure Lengend Snippet: IP pharmacokinetics of CLM296 in female NOD-SCID mice (A and B) CLM296 serum concentrations versus time curve and PK parameters in female NOD-SCID mice treated with a single dose of 4 mg/kg CLM296 administered via IP injection and collected at set time points. Each point represents an individual mouse ( n = 4 per time point). (C) CLM296 organ concentrations versus time curve in female NOD-SCID mice treated with a single dose of 4 mg/kg CLM296 administered via IP injection. Each point represents an individual mouse ( n = 4 per time point). (D) CLM296 serum concentrations versus time curve in female NOD-SCID mice bearing MDA-MB-231 tumors with ALDH1A3 OE were treated with a single dose of 4 mg/kg CLM296 administered via IP injection. Each point represents an individual mouse ( n = 4 per time point). (E) CLM296 tumor concentrations versus time curve in female NOD-SCID mice bearing MDA-MB-231 tumors with ALDH1A3 OE with a single dose of 4 mg/kg CLM296 administered via IP injection. Each point represents an individual mouse ( n = 4 per time point). (F) CLM296 organ concentrations versus time curve in female NOD-SCID mice bearing MDA-MB-231 tumors with ALDH1A3 OE treated with a single dose of 4 mg/kg CLM296 administered via IP injection. Each point represents an individual mouse ( n = 4 per time point).

Article Snippet: Anti-ALDH1A3 mouse IgG monoclonal primary antibody , Origene Technologies , Cat # CF502842 ; RRID; AB_3738370.

Techniques: Drug discovery, Injection

Journal: iScience

Article Title: Selective inhibition of ALDH1A3 impedes breast cancer growth and metastasis by blocking ALDH1A3-driven transcriptional programs

doi: 10.1016/j.isci.2026.114863

Figure Lengend Snippet: Oral pharmacokinetics of CLM296 in MDA-MB-231 ALDH1A3 OE tumor-bearing female NOD-SCID mice (A and B) CLM296 serum concentrations versus time curve in female NOD-SCID mice bearing MDA-MB-231 tumors with ALDH1A3 OE were treated with a single dose of 4 mg/kg CLM296 administered via oral gavage. Each point represents an individual mouse ( n = 4 per time point). (C) CLM296 tumor concentrations versus time curve in female NOD-SCID mice bearing MDA-MB-231 tumors with ALDH1A3 OE treated with a single dose of 4 mg/kg CLM296 administered via oral gavage. Each point represents an individual mouse ( n = 4 per time point, note that at early time points, a few of the tumor samples were below the limit of detection and so are not shown in the graph). (D) CLM296 organ concentrations versus time curve in female NOD-SCID mice bearing MDA-MB-231 tumors with ALDH1A3 OE treated with a single dose of 4 mg/kg CLM296 administered via oral gavage. Each point represents an individual mouse ( n = 4 per time point).

Article Snippet: Anti-ALDH1A3 mouse IgG monoclonal primary antibody , Origene Technologies , Cat # CF502842 ; RRID; AB_3738370.

Techniques: Drug discovery

Journal: iScience

Article Title: Selective inhibition of ALDH1A3 impedes breast cancer growth and metastasis by blocking ALDH1A3-driven transcriptional programs

doi: 10.1016/j.isci.2026.114863

Figure Lengend Snippet: CLM296 reduces ALDH1A3-mediated tumor growth and metastasis of MDA-MB-231 cells in NOD-SCID mice. (A) Tumor growth and weight of MDA-MB-231 vector control tumor-bearing female NOD-SCID mice treated with 0 mg/kg ( n = 9), 0.4 mg/kg CLM296 ( n = 10), or 4 mg/kg CLM296 ( n = 10), or ALDH1A3 OE tumor-bearing mice treated with 0 mg/kg ( n = 9), 0.4 mg/kg ( n = 10), or 4 mg/kg ( n = 9) CLM296. Treatment with CLM296 began once palpable tumors developed (indicated by the arrow, day 15) in the tumor volume plot, which demonstrates weekly caliper measurements. The tumor weight plot shows final tumor weight from the harvested tumors at endpoint. Tumor volume and weight significance were analyzed with two-way ANOVA with multiple comparisons post-test and p value < 0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. In the tumor volume graph, each point represents the mean of each group and error bars represent standard error of the mean (SEM). In the tumor weight graph, each point represents an individual mouse, and error bars represent SEM. (B) RNA extracted from the harvested tumors was analyzed via RT-qPCR for DHRS3 and RARβ expression. Significance was analyzed with two-way ANOVA with multiple comparisons post-test and p value < 0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. Each point represents an individual mouse and error bars represent SEM. (C) Body weights of the mice were measured weekly. Arrow indicates start of CLM296 IP injections. Each point represents the mean of each group and error bars represent SEM. (D) Serum creatinine and ALT of MDA-MB-231 vector control or ALDH1A3 OE tumor-bearing mice when treated daily with CLM296 via IP injection for 26 days. Significance was analyzed with two-way ANOVA with multiple comparisons post-test and p value < 0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. Each point represents an individual mouse and error bars represent SEM. (E) Transwell invasion assays were completed with vector control and ALDH1A3 OE MDA-MB-231 cells treated with or without 100 nM CLM296. FBS was used as a chemoattractant. Significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test and p value < 0.05 = ∗, <0.01 = ∗∗, <0.001 = ∗∗∗, <0.0001 = ∗∗∗∗, ns, not significant. Each point represents a separate n ( n = 6), and error bars represent SD. (F) Representative images of H&E-stained mouse lung sections from the second in vivo experiment taken from mice bearing MDA-MB-231 ALDH1A3 OE tumors treated with or without 4 mg/kg CLM296 ( n = 5 per group; 1 independent experiment). Arrows indicate metastatic lesions. (G) Quantification of MDA-MB-231 ALDH1A3 OE cells in the lung lobes of each mouse by RT-qPCR using human specific GAPDH primers. The horizontal line represents the limit of detection in the assay at 10 MDA-MB-231 per mouse lung lobe. Significance was analyzed with a one-way unpaired t test, and p value < 0.05 = ∗. Each point represents an individual mouse ( n = 9), and error bars represent SEM.

Article Snippet: Anti-ALDH1A3 mouse IgG monoclonal primary antibody , Origene Technologies , Cat # CF502842 ; RRID; AB_3738370.

Techniques: Plasmid Preparation, Control, Quantitative RT-PCR, Expressing, Injection, Staining, In Vivo